II_Animal Experimentation Techniques and Classical Genetics

Slide 3: The Origins of Genetics – G. Mendel

Key Points:

• Gregor Mendel, an Augustinian monk, is known as the father of genetics.

• He studied pea plants to understand how traits are inherited.

• Through his experiments, Mendel discovered that:

  • Traits are inherited in predictable patterns.

  • Each organism carries two alleles for each trait (one from each parent).

  • These alleles can be dominant or recessive.

Explanation of Visuals:

• A portrait of Mendel is shown to highlight his historical role.

• The slide briefly lists his work with traits such as flower color, seed shape, and pod color.

Glossary:

• Allele: A version of a gene.

• Dominant allele: The form that masks the other allele when present.

• Recessive allele: The form that is only expressed when two copies are present.

Key Takeaway:
Mendel’s work with pea plants laid the foundation for our understanding of how traits are passed from parents to offspring using dominant and recessive alleles.

Slide 4: Punnett Squares

Key Points:

• A Punnett square is a tool used to predict the outcome of genetic crosses.

• It shows how alleles from each parent combine in their offspring.

• Example cross: Aa (heterozygous) × Aa

  • Offspring possibilities:

    • 25% AA (homozygous dominant)

    • 50% Aa (heterozygous)

    • 25% aa (homozygous recessive)

Explanation of Visuals:

• The square grid shows four combinations of alleles.

• Parent alleles are placed on the top and side of the grid, with resulting genotypes filled inside.

Glossary:

• Punnett Square: A diagram that predicts the possible genetic makeup of offspring.

• Genotype: The genetic combination of alleles (e.g., Aa).

• Phenotype: The physical trait that results (e.g., yellow or green peas).

Key Takeaway:
Punnett squares help visualize the likelihood of inheriting different genotypes and phenotypes from parent crosses.

Slide 5: Naming the Results

Key Points:

• Hemizygous

  • Only one copy of the gene is present (no second allele).

  • Seen in X-chromosomal genes in males (males have only one X chromosome).

  • Also applies to:

    • Conventional transgenes when inserted only once.

    • Deletions of full genes or chromosomal regions.

• Heterozygous

  • Two different alleles are present on the two chromosomes.

  • Can arise from:

    • Point mutations

    • Variants

    • Insertions (e.g., Neo, Cre, GFP)

    • Small deletions

• Homozygous

  • Both chromosomes carry the same allele of a gene.

Explanation of Terms and Visuals:

• The slide is text-based, listing three genotype categories and their definitions.

• It compares hemizygous, heterozygous, and homozygous states in terms of gene copy number and genetic content.

• The focus is on recognizing these terms when analyzing genetic modification results in lab animals.

Glossary:

• Hemizygous: An organism has only one allele of a gene instead of two (e.g., males for X-linked genes).

• Heterozygous: Two different alleles of a gene are present.

• Homozygous: Two identical alleles of a gene are present.

• Transgene: An artificially inserted gene, often used in genetically modified organisms.

• Neo (Neomycin resistance), Cre (recombinase), GFP (Green Fluorescent Protein): Common genetic markers or tools used in gene engineering.

Key Takeaway:

This slide explains how to name genetic outcomes based on allele combinations:

• Hemizygous = one allele only,

• Heterozygous = two different alleles,

• Homozygous = two identical alleles.
  These terms help describe genetic modification results in research.

Slide 6: Dihybrid Cross – CD4cre and ZAP70lox

Key Points:

• This slide shows a dihybrid genetic cross involving two genes:

  • CD4-cre (a gene-modifying enzyme under the CD4 promoter)

  • ZAP70flox (a floxed version of the ZAP70 gene, used for conditional knockouts)

• The parental genotypes are both:

  • CD4⁽ᶜʳᵉ⁾ᵂᵀ / ZAP70⁽ᶠˡᵒˣ⁾ᵂᵀ

• The Punnett square shows all possible combinations of alleles that can result from this cross, using both:

  • Male gametes (sperm)

  • Female gametes (oocytes)
    Each row represents a combination coming from oocytes
    Each column represents a combination coming from sperm

• The colored grid shows all 16 genotype combinations of the offspring

• Genotype ratio:
  1:1:1:1:2:2:2:2:4
  (each cell type appears a specific number of times)

• Phenotype probabilities (based on genotype frequency):

  • 6.25% (1 out of 16)

  • 12.5% (2 out of 16)

  • 25% (4 out of 16)

Explanation of Visuals:

• The Punnett square is a 4×4 matrix representing all combinations of alleles from the two genes (CD4 and ZAP70) inherited from each parent.

• Columns = alleles from sperm

• Rows = alleles from oocytes (egg cells)

• Each box shows the resulting genotype of an offspring.

• The colors help visualize the frequency of each genotype.

Glossary:

• Dihybrid Cross: A genetic cross involving two genes.

• CD4-cre: A construct that expresses Cre recombinase under the CD4 promoter; used to manipulate genes in T cells.

• ZAP70flox: A gene flanked by loxP sites ("floxed"), allowing it to be deleted in cells expressing Cre.

• WT (Wild-Type): The normal, unmodified version of a gene.

• Gametes: Reproductive cells (sperm and oocytes) carrying one allele for each gene.

• Sperm/Oocytes: Male and female gametes contributing alleles to the offspring.

Key Takeaway:
This slide uses a Punnett square to model the expected offspring genotypes from a dihybrid cross involving CD4-cre and ZAP70flox alleles, showing how sperm and oocyte contributions result in a predictable ratio of genotypes..

Slide 7: Dihybrid Cross – CD4cre and ZAP70lox (continued)

Key Points:

• This slide continues the analysis of a dihybrid cross involving CD4cre and ZAP70lox alleles.

• Shows how genotypes segregate in the F2 generation.

• Each gene has dominant and floxed (lox) alleles, and combinations determine phenotypes.

Explanation of Visuals:

• Two Punnett squares (for each gene locus) show how CD4cre and ZAP70lox alleles combine.

• Boxes are color-coded to show genotypic combinations like +/+, flox/flox, cre/+, etc.

Glossary:

• CD4cre: A genetic construct used to drive gene expression in CD4+ T cells.

• ZAP70lox: A version of the ZAP70 gene that can be conditionally deleted (floxed).

• Floxed (lox): A gene flanked by loxP sites, allowing it to be removed by Cre recombinase.

Key Takeaway:
This slide visualizes how combinations of two genetically engineered alleles segregate in offspring using the dihybrid cross principle.

Slide 8: Crossover Breeding

Key Points:

• Crossover breeding is needed when your genes of interest are on the same chromosome, and you want to combine them in cis (on the same chromosome copy in the same animal).

• The diagram shows a female with KO1/KO1 and WT2/WT2 chromosomes crossed with a male carrying WT1/WT1 and KO2/KO2.

• After meiosis and recombination, you get offspring with recombinant chromosomes, such as:

  • WT1/KO1 and WT2/KO2 (highlighted in red) — this specific genotype contains a combination of both knockout alleles from different loci.

• This technique is used to bring together two modified alleles (e.g., KO1 and KO2) into one mouse.

Explanation of Visuals:

• Top section: Illustrates the parent genotypes.

  • Female: KO1/KO1 and WT2/WT2

  • Male: WT1/WT1 and KO2/KO2

• Middle section: Shows meiosis/crossover, where segments of chromosomes are exchanged.

• Bottom section: Visualizes possible offspring genotypes. The red circle highlights a recombinant mouse that has:

  • WT1 / KO1 on one chromosome

  • WT2 / KO2 on the other chromosome

• These recombinants are the desired result of crossover breeding.

Glossary:

• Crossover (Recombination): Exchange of genetic material between homologous chromosomes during meiosis.

• KO (Knockout): A gene that has been inactivated or deleted.

• WT (Wild-Type): The normal, unmodified version of a gene.

• Meiosis: The cell division process that produces gametes (sperm and eggs) with half the number of chromosomes.

• Recombination Hotspots: Regions of chromosomes with a high frequency of crossover events.

• Chromosome 1: Known to show increased recombination activity in females (about 1.2 times more than males).

Key Takeaway:
Crossover breeding allows the combination of different genetic modifications (like KO1 and KO2) from separate chromosomes into one animal by taking advantage of natural recombination during meiosis.

Slide 9: How Many Breeders Are Required?

Key Points:

• This slide raises an important planning question: how many breeding pairs are needed to obtain the desired number of genetically modified offspring?

• The answer depends on:

  • The probability of the desired genotype

  • The number of offspring per litter

  • The desired sample size for experiments

Explanation of Visuals:

• Text-only slide with the central question in blue: “HOW MANY BREEDERS ARE REQUIRED?”

• Prepares the viewer for the calculation method shown in the next slide.

Glossary:

• Breeder pair: A male and female used for mating in a genetic cross.

Key Takeaway:
Designing a breeding plan requires understanding genetic ratios and planning for enough litters to yield statistically meaningful numbers of target-genotype animals.

Slide 10: Normal Distribution of Outcome

Key Points:

• Breeding experiments (e.g. knockouts) result in random distributions of genotypes among offspring.

• Even if expected ratios are known, the actual outcome can vary due to chance.

• The diagram shows different genotypes resulting from combinations of alleles:

  • +/+ (wild-type)

  • +/− (heterozygous)

  • −/− (knockout)

• With two genes involved (e.g., two loci each with a possible KO), 56 genotype combinations are possible.

• A random draw (like a litter of 6 mice) can result in varying numbers of KO animals (e.g., 0, 1, 2, or 3 KOs).

• A histogram shows the probability distribution: most litters will produce 1–2 KOs, fewer produce 3 or more.

Explanation of Visuals:

• Top left: Genotype combinations from a Punnett square for a cross involving two genes (+/+, +/−, −/−).

• Middle: A pool of colored dots (representing different genotypes) is drawn to simulate offspring.

• Right: Offspring are grouped by number of KO alleles (0–3).

• Bar plot: Shows likelihood of getting a certain number of KO animals in a litter of 6.

• Bottom: Shows 56 possible combinations of genotypes.

• Example given: If you need 3 homozygous KO (−/−) animals, you would need to breed 21 offspring to have an 80% chance of success.

Glossary:

• KO (Knockout): A gene that has been inactivated or deleted.

• +/+: Both alleles are wild-type (normal).

• +/−: One allele is wild-type, one is knockout (heterozygous).

• −/−: Both alleles are knockout (homozygous knockout).

• Litter: A group of offspring born from one pregnancy.

• Normal distribution: A bell-shaped curve representing expected variation due to random chance.

Key Takeaway:
Even if expected Mendelian ratios are known, the actual outcome in a litter varies. To obtain a desired number of knockout animals, you often need to plan for more offspring than the minimum, due to the probabilistic nature of inheritance.

Slide 11: Litter Size

Key Points:

• This slide introduces the topic of litter size (number of pups born per birth).

• Litter size is a critical factor in planning the breeding of genetically modified mice.

• The average number of pups born influences how many breeding pairs are required to meet experimental goals.

Explanation of Visuals:

• The slide only shows the title “LITTER SIZE” in blue — it acts as a transition to the next section.

Glossary:

• Litter size: The number of offspring a female mouse gives birth to in a single litter.

• Breeding pair: A male and female mouse set up for reproduction.

Key Takeaway:
Litter size is a central parameter in mouse breeding experiments and must be considered during experimental planning.

Slide 12: Estimation of Litter Size

Key Points:

• Litter sizes vary and are subject to statistical variation, often following a Poisson distribution.

• Genetic background and experimental conditions can influence litter size.

• Breeding plans should include probabilistic modeling to account for this variability.

Explanation of Visuals:

• Several histograms show the probability distribution of litter sizes.

• The term Poisson is highlighted, referring to the relevant statistical distribution.

Glossary:

• Poisson distribution: A statistical distribution used to model rare or count-based events like litter sizes.

• Statistical variation: Natural differences in outcomes due to random biological factors.

Key Takeaway:
Litter size is variable and should be incorporated into breeding plans using statistical models like the Poisson distribution.

Slide 13: Fertility

Key Points:

• This slide introduces the concept of fertility (reproductive capability).

• Fertility greatly impacts how many breeding pairs will successfully produce offspring.

Explanation of Visuals:

• The slide contains only the title “FERTILITY” in blue and serves as a section divider.

Glossary:

• Fertility: The ability of a breeding pair to conceive and produce offspring.

• Reproductive success: The outcome of mating resulting in live offspring.

Key Takeaway:
Fertility is a key biological factor when designing effective breeding strategies.

Slide 14: Estimation of Fertility – Scenario 1

Key Points:

• Example showing how fertility rates affect offspring numbers.

• Not all breeding pairs produce offspring — only a proportion is successful.

• A histogram shows the distribution of litter sizes under 75% fertility.

Explanation of Visuals:

• Left: Breeding cages, some of which are empty (representing unsuccessful pairs).

• Right: A histogram showing how many offspring result from 75% fertility across breeding pairs.

Glossary:

• Fertility rate: The percentage of breeding pairs that successfully produce a litter.

• Simulation: A modeled representation of biological outcomes based on probability.

Key Takeaway:
Not all breeding pairs are fertile. Planning must include extra pairs to achieve a target number of animals.

Slide 15: Estimation of Fertility – Scenario 2

Key Points:

• Second scenario with a reduced fertility rate (~50%).

• Even more breeding pairs fail to produce offspring.

• The distribution shifts toward fewer offspring per breeding pair.

Explanation of Visuals:

• Left: More breeding cages with no offspring symbols.

• Right: Histogram reflects lower fertility, centered around fewer animals per pair.

Glossary:

• Reproductive efficiency: How effectively breeding pairs produce viable offspring.

• Simulation model: A predictive tool based on estimated biological parameters.

Key Takeaway:
Lower fertility results in more unproductive breeding and requires more pairs to reach the target cohort size.

Slide 16: Estimation of Fertility – Scenario 3

Key Points:

• Fertility is reduced further (~25%) — only 1 in 4 breeding pairs is successful.

• This dramatically increases the number of pairs needed to reach goals.

• Very wide variation in offspring numbers is observed.

Explanation of Visuals:

• Left: Most breeding cages are shown as unsuccessful.

• Right: Histogram shows many zero values and only a few litters with offspring.

Glossary:

• Production rate: The actual number of animals produced compared to the number of breeding pairs used.

• Variability: How much the actual results differ from expectations.

Key Takeaway:
As fertility decreases, breeding becomes more resource-intensive and unpredictable — more pairs, time, and animals are needed.

Slide 17: Reproduction Parameters I

Key Points:

• Table shows biological reproduction parameters for various mouse strains.

• Includes:

  • Fertility rate

  • Average litter size

  • Standard deviation

• These parameters vary between strains and must be considered in breeding plans.

Explanation of Visuals:

• The table compares mouse strains (e.g., C57BL/6J, BALB/c, FVB) by their reproductive characteristics.

• Clear differences in litter size and fertility rates are shown.

Glossary:

• Standard deviation: A measure of how much litter sizes vary from the average.

• Reproductive parameters: Measurable traits that describe how well a strain reproduces.

Key Takeaway:
Mouse strains differ in fertility and litter size, so planning must be tailored to the specific line being bred.

Slide 18: Cohort-Based Approach

Key Points:

• Introduction of cohort-based planning.

• Group size planning depends on:

  • Mendelian inheritance

  • Fertility

  • Litter size

• The goal is to obtain a defined group of animals for experiments.

Explanation of Visuals:

• Transition slide titled:
  “GROUP SIZE PLANNING AROUND MENDEL GENETICS, FERTILITY AND LITTER SIZE”

Glossary:

• Cohort: A defined group of animals of the same age or experimental assignment.

• Cohort-based planning: Working backward to calculate how many breeders are needed to reach a fixed number of desired animals.

Key Takeaway:
Cohort-based breeding calculates how many breeding pairs are needed to obtain a specific number of experimental animals.

Slide 19: Cohort-Based Breeding (Visual)

Key Points:

• Visualizes the planning model:

  • Breeding → Expected genotypes → Selection → Experimental animals

• Only a fraction of offspring have the desired genotype.

• Therefore, overproduction is needed to ensure enough suitable animals.

Explanation of Visuals:

• Flow diagram shows the breeding process through to selection of experiment-ready animals.

• Only offspring with the target genotype are included in the final cohort.

Glossary:

• Selection: Choosing animals with the desired genotype from a larger group.

• Overproduction: Deliberate breeding of more animals than needed to ensure enough with the correct traits.

Key Takeaway:
Cohort-based breeding requires intentional overproduction to ensure the target genotype is available in sufficient numbers.

Slide 20: Colony-Based Approach

Key Points:

• An alternative breeding strategy: colony-based breeding.

• Focus is on long-term production, not one-time cohorts.

• Planning is based on efficiency of the breeding line.

Explanation of Visuals:

• Title slide introducing:
  “GROUP SIZE PLANNING AROUND PRODUCTION EFFICIENCY INDEX”

Glossary:

• Colony: An ongoing breeding population that produces animals continuously.

• Production Efficiency Index (PEI): A measure of how effectively desired-genotype animals are generated.

Key Takeaway:
Colony-based breeding supports continuous animal production and is suited for long-term studies.

Slide 21: Colony-Based Breeding (Visual)

Key Points:

• Shows a continuous production model:

  • Multiple breeding pairs → Repeated litters → Ongoing animal supply

• Compared to cohort-based:

  • Higher total output

  • Better for ongoing experiments

Explanation of Visuals:

• Diagram illustrates the flow of regular offspring production from multiple breeders, feeding a steady pipeline of experimental animals.

Glossary:

• Ongoing production: Regular generation of animals over an extended period.

• Pipeline: The continuous supply of usable animals into experiments.

Key Takeaway:
Colony-based breeding ensures regular availability of animals and prevents shortages in long-term experimental setups.

Slide 22: Continuous Breeding of Large Cohorts

Key Points:

• This slide introduces the Production Efficiency Index (PEI), also known as the Productivity or Colony Index (CI).

• The PEI quantifies how efficiently a breeding colony produces usable offspring.

• It is calculated by dividing the number of weaned pups by the total number of breeding females over a specific time period (typically per week).

Explanation of Visuals:

• The slide is text-based and presents the PEI formula:

• $\frac{\text{Number of weaned pups}}{\text{Number of females} \times \text{Time (in weeks)}

• It references Festing and Peters, 1999 as the source.

Glossary:

• Production Efficiency Index (PEI): A measure of how many weaned pups are produced per female per week; used to assess breeding performance.

• Weaned pups: Offspring that have grown past the nursing stage and are no longer dependent on the mother.

• Colony Index (CI): Another term for PEI, often used in the context of ongoing colony maintenance.

• Continuous breeding: A breeding strategy aimed at regularly producing animals over time, as opposed to one-time production for a single cohort.

Key Takeaway:
The PEI helps evaluate the productivity of a breeding colony by standardizing the number of weaned pups per female per week, enabling comparison and optimization of breeding performance.

Slide 23: Production Efficiency Index – Example

Key Points:

• A table is shown with an example calculation of the Production Efficiency Index (PEI) for different breeding strategies.

• Input values include:

  • Fertility

  • Litter size

  • Genotype frequency

  • Number of litters

• The PEI allows for comparison between different strategies in terms of efficiency.

Explanation of Visuals:

• The table lists various breeding designs and their resulting PEI values.

• Efficiency varies depending on biological and genetic parameters.

Glossary:

• Production Efficiency Index (PEI): A calculated value showing how efficiently a breeding strategy produces the desired genotype.

Key Takeaway:
The PEI helps select the most resource-efficient breeding approach to obtain the required number of animals with the target genotype.

Slide 24: Impact of Fertility and Litter Size on Production

Key Points:

• Graphs show how fertility rate and litter size affect total production.

• Lower fertility or smaller litters require more breedings to reach the same goal.

• The effect is nonlinear—as fertility or litter size decreases, breeding demand increases rapidly.

Explanation of Visuals:

• Two line graphs:

  • Left: Shows output decline with lower litter sizes.

  • Right: Shows how reduced fertility impacts required breedings.

Glossary:

• Nonlinear relationship: A change in one variable leads to a disproportionately large effect in another.

Key Takeaway:
Small decreases in fertility or litter size greatly increase the number of breedings needed, underscoring the importance of accurate planning.

Slide 25: Gene Manipulation

Key Points:

• This slide introduces the topic of genetic modification.

• Title-only slide meant as a transition into methods and purposes of gene manipulation in mice.

Explanation of Visuals:

• Contains the title “Gene Manipulation” only, signaling a shift in the topic.

Glossary:

• Gene Manipulation: The deliberate alteration of an organism’s genetic material using molecular biology techniques.

Key Takeaway:
The next section will cover how and why genes are altered in laboratory animals like mice.

Slide 26: What Is the Genome?

Key Points:

• The genome consists of all the genetic material (DNA) in an organism.

  • It INCLUDES the MITOCHONDRIUM (DON’T Forget this!!!)

• DNA is organized into chromosomes, located in the nucleus.

• Each chromosome contains many genes.

Explanation of Visuals:

• Left: An image of karyotypes (chromosome pairs).

• Right: A graphic of a cell nucleus showing chromosomes condensed inside.

Glossary:

• Genome: The complete set of DNA (genes + non-coding regions) in an organism.

• Chromosome: A DNA molecule with part or all of the genetic material of an organism.

Key Takeaway:
The genome is the entire instruction manual of an organism, stored in chromosomes in each cell.

Slide 27: Why Is Genome Manipulation Done?

Key Points:

• Genetic manipulation is used to:

  • Study gene function

  • Model human diseases

  • Develop targeted therapies

  • Understand developmental biology

• Genetically modified mice are central to biomedical research.

Explanation of Visuals:

• A pie chart illustrates the main research areas where genetic manipulation is applied (e.g., cancer, immunology).

Glossary:

• Model organism: An animal used in experiments to understand biological processes relevant to humans.

Key Takeaway:
Genome manipulation allows scientists to simulate diseases and study gene function in detail using model organisms.

Slide 28: What Is Genome Manipulation?

Key Points:

• Shows the basic methods of gene manipulation:

  • Gene knock-in (adding a gene)

  • Gene knock-out (removing or inactivating a gene)

• Also shows conditional models using systems like Cre/lox, where gene changes happen only in specific tissues or time points.

Explanation of Visuals:

• Diagram compares wild-type, knock-out, and conditional knock-out genotypes.

• Arrows illustrate how DNA segments are modified or deleted.

Glossary:

• Knock-in: Adding a gene into a specific location in the genome.

• Knock-out: Deleting or disrupting a gene to stop its function.

• Conditional Knock-out: A gene is only removed in specific cells or under specific conditions (e.g., via CD4-Cre system).

Key Takeaway:
Gene manipulation enables researchers to precisely add, remove, or control gene expression to study biology and disease.

Slide 29: From Gene to Observable Phenotype

Key Points:

• The phenotype (what we see) is determined by genes and their expression.

• But one gene doesn’t have only 1 Phenotype necessarly.

• A gene codes for a protein, and that protein leads to a trait.

• Example: Different coat colors or body sizes in animals are driven by different genes and their resulting proteins.

Explanation of Visuals:

• Top row: Gene → mRNA → protein

• Bottom row: Protein → physical traits (like fur color or ear shape)

• Illustrates how genetic information is translated into a visible characteristic.

Glossary:

• Gene: A segment of DNA that codes for a protein.

• Phenotype: The observable trait (e.g., color, size) resulting from gene expression.

Key Takeaway:
A gene controls the production of proteins, and proteins ultimately shape the physical traits of an organism.

Slide 30: 25,000 BC – Selective Breeding

Key Points:

• Introduces the historical origin of selective breeding in early human civilization (~25,000 BC).

• Humans began choosing animals with desirable traits for reproduction.

Explanation of Visuals:

• The slide shows a prehistoric stone tool (symbolizing early human intervention).

• Title only; serves as a thematic transition to the history of breeding.

Glossary:

• Selective breeding: The intentional mating of individuals to produce offspring with preferred traits.

Key Takeaway:
Selective breeding began thousands of years ago as humans started shaping animal traits for domestication.

Slide 31: Dog Breeding

Key Points:

• Wolves were selectively bred into domestic dogs.

• Over generations, selective breeding produced a wide variety of dog breeds with specific appearances and behaviors.

• Modern dog breeds illustrate how targeted selection can dramatically reshape an animal species.

Explanation of Visuals:

• Sequence shows a wolf transforming into various dog breeds (e.g., bulldog, husky).

• A selection triangle represents narrowing the gene pool toward specific traits.

Glossary:

• Domestication: The process of adapting wild animals for human use through controlled breeding.

Key Takeaway:
Dog breeding is a clear example of how selective breeding transforms a wild species into a variety of domestic forms.

Slide 32: 1865 – Selective Breeding v2

Key Points:

• Marks the scientific shift in breeding during 1865, when more structured and purpose-driven methods were developed.

• Connects the timeline to Mendel’s discoveries that formalized genetics.

Explanation of Visuals:

• Slide shows only the title "Selective Breeding v2", signaling the next phase in breeding based on genetic understanding.

Glossary:

• Breeding v2: Refers to scientifically guided breeding, influenced by Mendelian principles.

Key Takeaway:
Selective breeding evolved from tradition to science after Mendel’s discoveries in 1865.

Slide 33: Breeding Laws – Gregor Mendel

Key Points:

• Gregor Mendel is the founder of classical genetics.

• He discovered the laws of inheritance using pea plants:

  1. Law of Segregation

  2. Law of Independent Assortment

• His experiments showed how traits are passed from parents to offspring in predictable ratios.

Explanation of Visuals:

• Portrait of Mendel on the left.

• Pea plant diagram on the right, showing how dominant and recessive traits segregate.

Glossary:

• Law of Segregation: Each parent contributes one of two alleles for a trait.

• Law of Independent Assortment: Traits are inherited independently if genes are on different chromosomes.

Key Takeaway:
Mendel's breeding experiments laid the foundation of genetic science and explained how traits are inherited.

Slide 34: 1902 – Starting Genetics

Key Points:

• Highlights the year 1902, when genetics emerged as a scientific discipline based on Mendel’s earlier work.

• Marks the beginning of using genetics to analyze and predict inheritance.

Explanation of Visuals:

• Title and a stylized image of a feather/quill (representing the start of formal documentation/science).

• No data or diagrams, just a transitional slide.

Glossary:

• Genetics: The scientific study of genes, inheritance, and variation in living organisms.

Key Takeaway:
Genetics began in the early 1900s, transforming breeding from observation to predictive science.

Slide 35: Eugene Müller – Mutations in the Fruitfly

Key Points:

• Introduces Eugène Müller, an early pioneer in studying mutations in fruit flies (Drosophila melanogaster).

• Fruit flies became a key model organism for genetic research.

• They have:

  • Short lifespans

  • Easily observable traits

  • High reproduction rate

Explanation of Visuals:

• Left: Images of wild-type and mutant fruit flies with visible phenotypic differences.

• Right: A photo of Eugène Müller and a timeline marking early discoveries.

Glossary:

• Mutation: A change in the DNA sequence of a gene.

• Model organism: A species extensively studied to understand biological processes.

Key Takeaway:
Fruit flies were among the first animals used to study inheritance and mutations, thanks to their experimental advantages.

Slide 36: Radiation-Induced Transmutations

Key Points:

• Radiation can cause mutations in DNA, leading to observable trait changes.

• Diagram shows how mutagenic agents were used to induce changes in fruit fly genetics.

• These experiments helped scientists map genes to chromosomes.

Explanation of Visuals:

• Diagram showing a series of crosses after irradiation.

• Arrows and labels track how mutations propagate across generations.

Glossary:

• Radiation-induced mutation: A change in DNA caused by exposure to radiation.

• Transmutation: An old term for induced mutation.

Key Takeaway:
Early scientists used radiation to intentionally cause mutations, helping to discover the locations and functions of genes.

Slide 37: 1982 – Conventional Transgenes

Key Points:

• Marks the year 1982, when transgenic animals (with foreign genes) became possible.

• The technique allowed scientists to insert new genes into the genome.

Explanation of Visuals:

• Title-only slide with a symbolic image of a DNA-injecting needle (representing microinjection).

Glossary:

• Transgene: A gene that is transferred from one organism into the genome of another.

• Conventional transgenic model: A model where a foreign gene is inserted randomly into the genome.

Key Takeaway:
The introduction of transgenes in 1982 was a turning point in experimental genetics, enabling the creation of genetically modified animals.

Slide 38: 1st Functional Transgenic Mouse

Key Points:

• This slide shows the first successful creation of a functional transgenic mouse.

• Scientists inserted a rat growth hormone gene into a mouse.

• The result: mice grew significantly larger in size than normal.

• This was a major milestone in genetic engineering and transgenesis.

Explanation of Visuals:

• Top photo: Likely shows the two main scientists (Palmiter and Brinster) involved in the original experiment.

• Bottom left: The famous Nature magazine cover featuring a large transgenic mouse next to a normal-sized one.

• Right side: A growth curve plot showing that transgenic mice (labeled as "hGH-x") gained more weight over time than controls.

Glossary:

• Transgenic mouse: A mouse that carries a gene introduced from another species (in this case, a rat).

• Growth hormone (GH): A hormone that promotes growth and development in animals.

• Functional transgene: A gene that is not only inserted but also actively expressed in the host organism.

• hGH (human growth hormone): The label used for the inserted hormone gene in the study.

Key Takeaway:
The first functional transgenic mouse was created by inserting a rat growth hormone gene, resulting in increased body size — demonstrating that foreign genes can be functionally expressed in mammals.

Slide 39: Transgenes

Key Points:

• This slide explains how transgenes are inserted into the genome and how they behave.

• A transgene vector typically includes a promoter and a cDNA (gene of interest).

• Integration into the genome occurs randomly.

• Frequently, multiple copies of the transgene insert together — forming concatamers.

• This can lead to uncontrolled expression or instability of the inserted gene.

Explanation of Visuals:

• Top: A simple vector with a promoter and cDNA ready for insertion.

• Middle: Shows one copy integrating randomly into the genome (blue box: "random integration").

• Bottom: Shows multiple insertions in tandem (concatamers), which often happens during this process.

• Arrows indicate the direction of transcription for the inserted gene(s).

Glossary:

• Promoter: A DNA sequence that controls when and where a gene is expressed.

• cDNA: Complementary DNA, often used to represent coding sequences without introns.

• Random integration: The insertion of a transgene at an unpredictable site in the genome.

• Concatamer: A sequence of repeated transgene units joined end-to-end.

• Transgene vector: A DNA construct designed to deliver and express a gene in a host organism.

Key Takeaway:
Transgenes integrate randomly into the genome, often as multiple copies. This randomness can affect expression levels and must be considered when designing transgenic models.

Slide 40: Problems with Conventional Transgenes

Key Points:

• Lists major issues with traditional transgenic models:

  • Position effects

  • Uncontrolled copy number

  • Unpredictable expression

• These issues led to the development of targeted gene editing approaches like Cre/lox or CRISPR.

Explanation of Visuals:

• Left: Cartoon showing variable transgene integration.

• Right: Arrows show gene expression differences caused by random locations.

Glossary:

• Copy number variation: Number of copies of the inserted gene may differ between animals.

• Gene targeting: More precise method to insert genes at a known location.

Key Takeaway:
Because of variability in expression and location, conventional transgenes are being replaced by more targeted, predictable genetic tools.

Slide 41: Transgenesis by Pronuclear Microinjection

Key Points:

• Pronuclear injection is a method to create transgenic animals.

• DNA is injected into one of the two pronuclei of a fertilized egg.

• The foreign gene integrates randomly into the genome.

• This technique is widely used in mice to study gene function.

Explanation of Visuals:

• Image of a needle injecting DNA into the pronucleus of a zygote.

• Labels identify the pronuclei and the injection path.

Glossary:

• Pronucleus: The nucleus of a sperm or egg cell before they fuse during fertilization.

• Microinjection: A precise technique to deliver DNA directly into cells using a fine needle.

Key Takeaway:
Pronuclear microinjection is a foundational method in transgenic research, enabling direct introduction of DNA into early embryos.

Slide 42: Inducible Transgene Expression – Tet-Off/On Systems

Key Points:

• This slide explains two types of inducible transgene expression systems: Tet-Off and Tet-On.

• These systems allow researchers to control gene expression using doxycycline, an antibiotic.

Tet-Off System:

• The transcriptional activator tTA binds to the TRE promoter to activate the transgene — only when doxycycline is absent.

• When doxycycline is present, it binds to tTA and prevents it from binding DNA → transgene is turned off.

• So: Remove doxycycline → transgene ON.

Tet-On System:

• Uses a modified activator rtTA, which requires doxycycline to function.

• Only when doxycycline is added does rtTA bind to the TRE promoter and activate transgene expression.

• So: Add doxycycline → transgene ON.

Explanation of Visuals:

• Left diagram: Tet-Off system — doxycycline blocks tTA from binding → removal of doxycycline activates transgene.

• Right diagram: Tet-On system — rtTA needs doxycycline to bind the TRE promoter → adding doxycycline activates transgene.

• Both show how transcription of the transgene is regulated by doxycycline.

Glossary:

• tTA (tetracycline-controlled transactivator): A protein that binds DNA to activate gene expression in the absence of doxycycline (Tet-Off).

• rtTA (reverse tTA): A modified form that activates gene expression only when doxycycline is present (Tet-On).

• TRE (Tetracycline Response Element): A DNA sequence that tTA or rtTA binds to in order to control transcription of the transgene.

• Doxycycline: A tetracycline antibiotic used here to control gene expression in a reversible and dose-dependent manner.

• Inducible expression: A system where gene activity can be switched on or off by an external molecule (here, doxycycline).

Key Takeaway:
Tet-Off and Tet-On systems enable precise temporal control of transgene expression using doxycycline — a powerful tool for studying gene function in living organisms.

Slide 43: RT of Transgenic Models for Monogenic Diseases

Key Points:

• Transgenic mice are valuable for studying monogenic diseases (caused by mutations in a single gene).

• Example shown: a mouse model mimicking human obesity caused by a specific mutation.

• Disease phenotypes can be studied in a controlled setting.

• What is it good or benefical for to induce a trnasgene?

  • in type of animal welfare, you only induce the trsngen when you want to study the disease

  • so mice are not constantly in the diseased state.

Explanation of Visuals:

• Graph showing body weight data comparing wild-type and mutant mice.

• Photos of mice clearly showing size differences due to genetic mutation.

Glossary:

• Monogenic disease: A condition caused by a mutation in a single gene (e.g., cystic fibrosis, Huntington’s).

• Phenotype: Observable physical or biological characteristics.

Key Takeaway:
Transgenic mice allow researchers to model human monogenic diseases and study the resulting phenotypes in detail.

Slide 44: Cell Ablation by Diphtheriatoxin

Key Points:

• This slide explains a method to selectively destroy cells using diphtheria toxin (DT).

• The system relies on expressing the diphtheria toxin receptor (DTR) specifically in the target cells.

• Mice normally don’t express DTR, so only genetically modified (DTR+) cells are affected.

• When diphtheria toxin is administered, only DTR-expressing cells bind the toxin and undergo cell death.

Mechanism:

• Diphtheria toxin binds to DTR on the surface of target cells.

• The toxin is internalized, and its A subunit is released into the cytoplasm.

• It blocks protein synthesis by inactivating elongation factor 2 (EF2) via ADP-ribosylation.

• This leads to cell death, effectively ablating the target cell population.

Explanation of Visuals:

• Left diagram: Shows a DTR-expressing cell that binds diphtheria toxin and is eliminated (crossed out).

• Right diagram: Molecular mechanism of DT action:

  • Entry via endocytosis

  • Proteolytic cleavage and translocation of active subunit

  • EF2 inhibition → halt in protein synthesis

Glossary:

• Diphtheria toxin (DT): A potent bacterial toxin that blocks protein synthesis in susceptible cells.

• DTR (diphtheria toxin receptor): A receptor not naturally present in mouse cells, but introduced genetically for selective targeting.

• Cell ablation: The targeted destruction of specific cells.

• EF2 (elongation factor 2): A key molecule required for protein synthesis during translation.

• ADP-ribosylation: A chemical modification that inactivates EF2 in this context.

Key Takeaway:
By introducing the diphtheria toxin receptor into specific cells, researchers can selectively eliminate those cells using diphtheria toxin — a precise tool for studying cell function through targeted ablation.

Slide 45: Channels and More

Key Points:

• This slide presents two types of genetic tools used for modulating cell function:

  • Channelrhodopsin (ChR) and Halorhodopsin (light-controlled ion channels)

  • DREADDs (Designer Receptors Exclusively Activated by Designer Drugs)

Channelrhodopsin / Halorhodopsin:

• Channelrhodopsins are activated by light and allow Na⁺ ions to enter cells, causing activation (e.g. neuron firing).

• Halorhodopsins are activated by light and allow Cl⁻ ions to enter, causing inhibition of cellular activity.

DREADDs:

• These are modified G-protein coupled receptors that respond only to synthetic ligands, not natural ones.

• Different types of DREADDs (e.g., hM3Dq, hM4Di, KORD) activate or inhibit different signaling pathways (e.g., Gq, Gi, Gs, K⁺ channels).

• DREADDs allow remote control of cell behavior through administration of designer drugs like CNO or Salvinorin B.

Explanation of Visuals:

• Left: Diagrams show how light triggers channel opening for Na⁺ or Cl⁻ flow via channelrhodopsins or halorhodopsins.

• Right: Shows four DREADD types, each with a specific signaling target and ligand.

• Text below: Defines DREADDs as receptors engineered to respond only to synthetic ligands.

Glossary:

• Channelrhodopsin (ChR): A light-gated ion channel that allows sodium ions into the cell, used in optogenetics.

• Halorhodopsin: A light-activated chloride pump that inhibits neurons.

• DREADD: Engineered receptor activated by synthetic, non-native drugs.

• CNO (Clozapine-N-oxide), Salvinorin B: Example ligands that activate DREADDs.

• Optogenetics: A technique using light to control genetically modified cells.

Key Takeaway:
Channelrhodopsins and DREADDs are powerful tools to control cell activity with light or designer drugs, enabling precise functional studies in living systems.

Slide 46: Inducible Protein Degradation – Auxin-Inducible Degron

Key Points:

• This slide introduces a system for conditional protein degradation using plant-derived mechanisms.

• A protein of interest is tagged with the mini-AID degron sequence.

• In cells that express the plant protein OsTIR1 (from Oryza sativa), auxin addition triggers protein degradation.

Mechanism:

• OsTIR1 forms part of the SCF E3 ubiquitin ligase complex.

• When auxin is present, OsTIR1 binds to the AID tag on the target protein.

• The complex then labels the target protein for degradation by the proteasome.

Explanation of Visuals:

• Diagram shows:

  • Target protein with mini-AID tag.

  • Interaction with OsTIR1 and SCF complex upon auxin addition.

  • Resulting protein degradation through the proteasome.

Glossary:

• Auxin: A plant hormone used here to trigger degradation.

• mini-AID (auxin-inducible degron): A short sequence added to a protein that marks it for degradation in presence of auxin.

• SCF complex: A multi-protein E3 ligase complex that tags proteins for degradation.

• Proteasome: The cellular machinery that degrades unwanted or misfolded proteins.

• OsTIR1: A plant F-box protein that recognizes the AID tag when auxin is present.

Key Takeaway:
The auxin-inducible degron system allows rapid and reversible control of protein levels by degrading specifically tagged proteins in the presence of auxin.

Slide 47: 1987 – Gene Targeting

Key Points:

• Marks the scientific milestone of 1987, when gene targeting via homologous recombination became possible.

• This allowed precise editing of specific genes in mice.

• Laid the foundation for knock-out and knock-in mouse models.

Explanation of Visuals:

• Title slide with symbolic DNA sequence and surgical blade, representing precise genetic editing.

Glossary:

• Gene targeting: Technique for modifying a specific DNA sequence in its natural chromosomal location.

Key Takeaway:
The development of gene targeting revolutionized genetics by enabling precise, targeted modifications in the genome.

Slide 48: Embryonic Stem Cells

Key Points:

• Gene targeting is performed using embryonic stem (ES) cells, which can differentiate into any tissue.

• These cells are genetically modified in vitro and then injected into blastocysts.

• Modified mice are created by breeding chimeras derived from these blastocysts.

Explanation of Visuals:

• Diagram shows isolation and culture of ES cells, their modification, and reintroduction into embryos.

• Includes photo of chimeric mouse.

Glossary:

• Embryonic stem cells (ES cells): Pluripotent cells derived from early embryos, capable of becoming any cell type.

• Chimera: An animal composed of genetically distinct cells from two zygotes.

Key Takeaway:
ES cells are key tools in gene targeting, enabling researchers to engineer specific genetic changes in whole animals.

Slide 49: Homologous Recombination

Key Points:

• Homologous recombination is the core mechanism in gene targeting.

• A targeting vector with homology arms matches the desired gene location.

• Allows precise gene replacement or disruption.

Explanation of Visuals:

• DNA schematic showing homology regions and insertion of a selection cassette via recombination.

Glossary:

• Homologous recombination: Exchange of genetic material between similar or identical DNA sequences.

• Targeting vector: A piece of DNA designed to insert a specific sequence at a desired genomic location.

Key Takeaway:
Homologous recombination enables precise modifications at specific genomic sites using engineered DNA constructs.

Slide 50: Gene Targeting

Key Points:

• This slide shows the workflow of gene targeting in mouse embryonic stem (ES) cells.

• Gene targeting allows the precise modification of specific genes, for example, to generate knockouts or insert specific sequences.

• The method uses a targeting vector, electroporation, selection, and screening to identify correctly modified ES cells.

Explanation of Visuals and Step-by-Step Procedure:

1. Start with ES Cell Colonies on Feeder Layer

   • Embryonic stem (ES) cells are grown on a layer of embryonic fibroblasts (EF) that support their growth.

2. Electroporation of Targeting Vector

   • The targeting DNA vector is introduced into ES cells using electroporation (an electrical pulse creates pores in the membrane so DNA can enter).

3. Selection with G418

   • Only cells that integrated the targeting vector survive because the vector carries a neomycin resistance gene (Neo⁺).

   • G418 is an antibiotic that kills unmodified cells.

4. Pick Colonies into 96-Well Plate and Freeze

   • Individual resistant ES cell colonies are picked and transferred into 96-well plates.

   • Aliquots are frozen for storage while others are used for screening.

5. Lyse Cells and Screen for Correct Clones

   • Cells are lysed to extract DNA.

   • Colonies are screened using Southern blot or PCR to determine which ones have the correct gene targeting (e.g., homologous recombination at the desired locus).

6. Thaw and Amplify Correct Clones

   • Clones with successful targeting are thawed (aufgetaut) from frozen stock.

   • These correct clones are expanded and used for further steps (e.g., blastocyst injection to make chimeric mice).

Glossary:

• ES cells (Embryonic Stem cells): Pluripotent cells derived from the early embryo that can be genetically modified and used to generate genetically modified mice.

• Targeting vector: A DNA construct designed to introduce specific genetic changes into the genome.

• Electroporation: A technique using electrical pulses to introduce DNA into cells.

• G418: An antibiotic used for selection of genetically modified cells carrying a neomycin resistance gene.

• Southern blot / PCR: Laboratory methods used to confirm that the gene modification occurred at the correct genomic location.

• Feeder layer: A layer of cells (e.g., fibroblasts) used to support the growth of ES cells.

Key Takeaway:
Gene targeting in ES cells involves introducing a targeting vector by electroporation, selecting resistant clones with G418, screening for correct genomic integration, and expanding validated clones for generating genetically modified animals.

EXTRA INPUT

🧪 What is a Southern Blot?

A Southern blot is a laboratory method used to detect a specific DNA sequence in a sample.
It is often used in gene targeting experiments to check whether a genetic modification (like a knockout or insertion) was successfully integrated into the genome.

🔬 Step-by-Step: How a Southern Blot Works

1. DNA Extraction

   • DNA is isolated from the cells (e.g., targeted ES cells).

2. DNA Digestion with Restriction Enzymes

   • The DNA is cut into fragments using restriction enzymes that cut at specific sequences.

   • These enzymes are chosen to produce different fragment sizes depending on whether the gene is modified or not.

3. Gel Electrophoresis

   • The DNA fragments are separated on an agarose gel by size.

   • Small fragments move faster; large fragments move slower.

4. DNA Denaturation

   • The gel is treated with a chemical (usually an alkaline solution) to denature the DNA (make it single-stranded).

5. Transfer to a Membrane (Blotting)

   • The single-stranded DNA fragments are transferred from the gel to a nitrocellulose or nylon membrane.

   • This is done by capillary action, vacuum, or electric current (blotting step).

6. Hybridization with a Probe

   • A radioactive or fluorescent DNA probe is added to the membrane.

   • The probe is complementary to the DNA sequence you want to detect.

   • The probe binds (hybridizes) only to its matching sequence.

7. Detection

   • After washing off unbound probes, the membrane is exposed to X-ray film or scanned with a detector.

   • Bands appear where the probe bound to the DNA — you can now see the size and presence of the target fragment.

🧬 Why is this useful in gene targeting?

• When you insert a targeting vector, it changes the size of the DNA fragments cut by the restriction enzyme.

• By comparing:

  • Wild-type band size (unchanged gene)

  • Targeted band size (with insertion or recombination),

• You can confirm whether homologous recombination happened at the correct spot.

🧠 Glossary:

• Restriction enzyme: Cuts DNA at specific sequences.

• Probe: A short, labeled DNA strand that binds to your target sequence.

• Hybridization: The process of the probe binding to its matching DNA.

• Blotting: Transferring DNA from gel to membrane for easier handling.

✅ Key Takeaway:

A Southern blot lets you confirm whether your genetic modification occurred exactly where and how you intended by detecting DNA fragment sizes after cutting and probing — it’s a gold-standard technique in verifying gene targeting.

n Blot

Slide 51: Selection of Recombinants (Neo+/TK–)

Key Points:

• Selection process isolates only the correctly recombined ES cells.

• Neo+/TK– cells indicate homologous recombination (Neo cassette inserted, TK excluded).

• These cells are expanded and injected into embryos.

Explanation of Visuals:

• Diagram compares outcomes of homologous vs. random integration and how selection enriches the desired outcome.

Glossary:

• Neo+/TK–: Cells that are resistant to neomycin (Neo+) and sensitive to ganciclovir (TK–), confirming correct targeting.

Key Takeaway:
Selection using Neo and TK markers efficiently distinguishes correctly targeted cells from incorrect ones.

Slide 52: Selection of Recombinants (Agarose Gel)

Key Points:

• Final confirmation of recombination is done using PCR or Southern blot.

• Agarose gel electrophoresis shows distinct bands for:

  • Wild-type

  • Targeted allele

• Used to confirm correct integration at the DNA level.

Explanation of Visuals:

• Diagram showing gel electrophoresis result with labeled lanes for targeted and untargeted alleles.

Glossary:

• Agarose gel electrophoresis: A method to separate DNA fragments by size for visualization.

• Southern blot: A technique for detecting specific DNA sequences after gel separation.

Key Takeaway:
Molecular techniques like gel electrophoresis confirm successful gene targeting before proceeding with mouse generation.

Slide 53: Making Targeted Mice

Key Points:

• This slide summarizes the entire process of generating a genetically modified (targeted) mouse.

• The key steps include:

  • Genetic modification of embryonic stem (ES) cells using a targeting vector.

  • Injection of modified ES cells into early-stage mouse embryos (blastocysts).

  • Transfer of injected embryos into a surrogate (foster) mother.

  • Birth of chimeric mice, which contain a mix of modified and unmodified cells.

  • Breeding of chimeras with wild-type mice to check for germline transmission (whether the genetic change is inherited).

  • Final breeding to obtain mice that are homozygous knockouts (both gene copies modified) for functional studies.

Explanation of Visuals:

• Top part: A step-by-step diagram showing:

  • Injection of ES cells into a blastocyst.

  • Implantation of the blastocyst into a surrogate female.

• Bottom part:

  • Photos showing a chimeric mouse (patchy coat indicating mixed genetic origin).

  • A fully targeted mouse, where the genetic modification has been inherited and is present in all cells.

Glossary (Extended):

• Embryonic Stem (ES) cells: Pluripotent cells derived from early mouse embryos; they can contribute to all tissues, including the germline.

• Targeting vector: A DNA construct used to introduce a specific genetic change into the genome via homologous recombination.

• Blastocyst: A pre-implantation stage embryo into which modified ES cells are injected.

• Surrogate mother: A foster female mouse that carries and gives birth to the embryos after injection.

• Chimera: A mouse composed of both host blastocyst cells and injected ES cells. Coat color differences often reveal the degree of chimerism.

• Germline transmission: Occurs when the modified ES cells contribute to egg or sperm cells, allowing the genetic change to be passed on to offspring.

• Homozygous knock-out: A mouse in which both alleles (gene copies from mother and father) are inactivated. Used for studying the function of the gene by observing the effects of its complete loss.

Key Takeaway:
Creating a genetically modified mouse involves targeted gene editing in stem cells, embryo manipulation, and careful breeding. The goal is to achieve heritable and precise gene knockouts for functional research in vivo

🧬 After Gene Targeting: What Happens?

Once you've successfully introduced your genetic modification into embryonic stem (ES) cells and injected those into a blastocyst, the resulting mouse that is born is a chimera.

🧪 Step-by-Step: From Gene Targeting to Knockout Mouse

1. Inject modified ES cells into a blastocyst

• The blastocyst has its own cells (normal).

• You inject genetically modified ES cells (with your targeting vector).

• These mix → leading to a chimera (mixed origin).

2. Chimera is born

• The chimera contains some cells from the modified ES cells, and some from the host blastocyst.

• Some chimeras might have germ cells (sperm or egg) derived from the modified ES cells, and some might not.

3. Breed the chimera with a wild-type mouse

• You pair this chimera with a normal (non-mutated) mouse.

• Your goal: see if the mutation made it into the germline (i.e., sperm or egg).

• You look at the offspring of this mating.

4. Offspring = heterozygous for the mutation

• These mice now have:

  • One normal gene (from the wild-type parent)

  • One targeted (mutated or knocked-out) gene (from the chimera parent)

• These mice are called heterozygous (e.g., +/−).

5. Breed heterozygous offspring with each other

• When you mate two heterozygous mice (+/− × +/−):

  • 25% of the next generation will be homozygous knockout (−/−)

  • 50% will be heterozygous (+/−)

  • 25% will be wild-type (+/+)

🔁 Summary in One Sentence:

After generating a chimera, you cross it with a wild-type mouse to test for germline transmission; if successful, the first generation offspring carry the mutation, and the second generation (from breeding heterozygotes) can produce homozygous knockout mice..

Slide 54: Proof of Gene Targeting

Key Points:

• Gene targeting must be verified before concluding success.

• Common methods:

  • Southern blot to detect DNA fragments

  • Check correct insertion using probe hybridization

• Bands on the gel differ between wild-type and targeted allele.

Explanation of Visuals:

• Left: Image of a Southern blot gel.

• Right: Diagram showing expected band sizes for successful targeting.

Glossary:

• Probe: A DNA fragment used to detect complementary sequences.

• Southern blot: A technique for detecting specific DNA sequences in DNA samples.

Key Takeaway:
Targeted gene insertion is confirmed by molecular analysis, ensuring the genetic modification occurred precisely.

Slide 55: Knock-out Programs

Key Points:

• Several global initiatives aim to systematically knock out every gene in the mouse genome.

• Examples:

  • KOMP: Knockout Mouse Project (USA)

  • EUCOMM: European Conditional Mouse Mutagenesis Program

  • NorCOMM: Canadian effort

• The goal is to study gene function on a genome-wide scale.

Explanation of Visuals:

• Bullet point list of programs, timelines, and acronyms.

Glossary:

• Knock-out mouse: A mouse in which a specific gene has been inactivated.

Key Takeaway:
International knock-out initiatives have made thousands of gene-deficient mice available for biomedical research.

Slide 56: Knock-Outs: Limitations

Key Points:

• Global knock-outs may lead to:

  • Embryonic lethality

  • Compensatory effects

  • Widespread side effects

• Solution: use conditional knock-out strategies to control when and where a gene is inactivated.

Explanation of Visuals:

• Bullet points emphasize why full knock-outs can be problematic.

• Suggestion to use Cre/lox system.

Glossary:

• Embryonic lethality: Death of the embryo due to loss of an essential gene.

• Conditional knock-out: A strategy that allows gene deletion in a specific tissue or at a specific time.

• Cre recombinase: Enzyme that cuts DNA between two loxP sites.

• loxP site: Short DNA tag used to guide Cre where to cut.

• Cas9: DNA-cutting protein used in CRISPR.

• Guide RNA (gRNA): Custom RNA that tells Cas9 where to cut.

Key Takeaway:
Knock-outs can cause unintended effects; conditional approaches offer more precision in genetic research.

Slide 57: How to Overcome It! – The Development of Conditional Mutagenesis

Key Points:

• This slide introduces conditional mutagenesis as a solution to limitations of traditional (global) knockout models.

• Conditional systems allow more precise gene targeting — in terms of when and where the gene is mutated.

• Three major approaches are listed:

  • Cell type–specific mutagenesis: Mutation occurs only in certain cell types (e.g., liver cells, neurons).

  • Inducible mutagenesis: Mutation is triggered at a specific time point, e.g., by adding a drug like tamoxifen or doxycycline.

  • Cell type–specific inducible mutagenesis: Combines both — gene is mutated in specific cells and only upon induction.

Explanation of Visuals:

• This is a transition slide summarizing the evolution toward more refined genetic models.

• The title "How to overcome it!" implies that earlier issues (e.g., embryonic lethality, broad off-target effects) can be addressed by conditional systems.

Glossary:

• Conditional mutagenesis: A strategy to inactivate or alter genes only under specific conditions, such as in certain tissues or at certain times.

• Cell type–specific: Gene targeting limited to selected tissues or organs (e.g., using promoters like Alb-Cre for liver or Nestin-Cre for neurons).

• Inducible: Gene modification occurs only after administration of an inducing agent (e.g., doxycycline, tamoxifen).

• Cre/loxP system: A common tool for conditional mutagenesis using Cre recombinase to cut DNA at loxP sites.

• Tamoxifen: A drug often used to activate Cre-ER fusion proteins in inducible systems.

• Doxycycline: An antibiotic used to control gene expression in Tet-On/Tet-Off systems.

Key Takeaway:
Conditional mutagenesis — whether cell type–specific, inducible, or both — allows precise control over gene function, helping overcome limitations like embryonic lethality or systemic effects in conventional knockout models.

Slide 58: Nobel Ceremony 2007

Key Points:

• Recognition of pioneering work in gene targeting.

• Nobel Prize in Physiology or Medicine 2007 awarded to:

  • Mario Capecchi

  • Martin Evans

  • Oliver Smithies

• Honored for their discoveries enabling targeted gene modification in mice.

Explanation of Visuals:

• Top: Nobel medal image.

• Bottom: Photos of the three laureates.

Glossary:

• Targeted gene modification: Inserting or deleting genes at specific locations.

Key Takeaway:
The ability to precisely modify mouse genes was so revolutionary that it earned a Nobel Prize in 2007.

Slide 59: 1994 – CRE/LOX

Key Points:

• Marks the 1994 implementation of the Cre/lox system in mice.

• A key development for conditional gene editing.

Explanation of Visuals:

• Title slide with image of a recombination “scalpel,” symbolizing precision.

Glossary:

• Cre/lox system: A method allowing gene deletion, activation, or inversion at specific DNA sites.

Key Takeaway:
The Cre/lox system provided a breakthrough for tissue-specific and time-controlled gene modifications.

Slide 60: The Cre/loxP System

Key Points:

• This slide explains the Cre/loxP system, a widely used genetic tool for site-specific DNA recombination.

• Cre recombinase is an enzyme derived from bacteriophage P1 that recognizes and acts on loxP sites.

• Depending on the orientation of loxP sites, Cre can cause:

  • Deletion of the DNA segment between them (if loxP sites are in the same direction).

  • Inversion of the DNA segment (if loxP sites are in opposite directions).

• This system is fundamental to conditional gene knockouts and inducible genetic modifications in mice.

Explanation of Visuals:

• Top: The sequence of the loxP site is shown, with the core region (in blue) being the asymmetric site that determines recombination direction.

• Left diagram: Two loxP sites flank exon 4 in the same direction → Cre-mediated deletion removes exon 4.

• Right diagram: Two loxP sites flank exon 4 in opposite directions → Cre-mediated inversion flips exon 4.

• Bottom (red text): Lists alternative recombination systems used in similar strategies:

  • Flp/FRT, Dre/rox, Vika/vox

Glossary:

• Cre recombinase: An enzyme that catalyzes recombination between two loxP sites.

• loxP site: A 34-base pair DNA sequence recognized by Cre; composed of two 13 bp palindromic arms and an 8 bp asymmetric core (which determines directionality).

• Deletion: Removal of a DNA segment — commonly used for gene knockouts.

• Inversion: Flipping the DNA segment between two oppositely oriented loxP sites.

• Conditional knockout: A gene deletion restricted to specific tissues or developmental stages.

• Flp/FRT system: Similar to Cre/loxP, but uses the Flp recombinase and FRT sites.

• Dre/rox and Vika/vox: Alternative site-specific recombinase systems used to avoid cross-talk with Cre/loxP.

Key Takeaway:
The Cre/loxP system enables precise genetic manipulation — including gene deletion or inversion — by targeting specific DNA sequences, making it a cornerstone of conditional mutagenesis in genetic mouse models.

Slide 61: Conditional Targeting (Tissue-Specific Deletion)

Key Points:

• Describes how Cre/loxP is used for tissue-specific gene deletion.

• Strategy:

  • Create a floxed mouse (gene flanked by loxP).

  • Cross with a Cre-expressing line (Cre active in specific tissues).

  • Only cells expressing Cre will undergo gene deletion.

Explanation of Visuals:

• Diagrams of breeding strategy: floxed gene + Cre leads to deletion in targeted cells.

• Gene deletion is restricted to tissues where Cre is expressed (e.g., liver, brain).

Glossary:

• Floxed gene: A gene flanked by loxP sites.

• Tissue-specific promoter: A promoter that activates gene expression only in selected cell types.

Key Takeaway:
Conditional targeting enables researchers to study gene function in specific tissues without affecting the whole organism.

Slide 62: First and Cell Type-Specific Knockout

Key Points:

• Shows validation of Cre/lox function in specific cell types.

• Western blot and PCR confirm gene deletion only in tissues where Cre is active.

• Helps verify tissue specificity and efficiency.

Explanation of Visuals:

• Left: PCR strategy to detect deleted allele.

• Right: Gel images show knockout only in Cre+ tissues.

Glossary:

• Western blot: Technique to detect specific proteins.

• Cell type-specific knockout: Gene is inactivated only in targeted cell types.

Key Takeaway:
Conditional knockouts can be experimentally confirmed using molecular methods to ensure accuracy.

Slide 63: Conditional Mutagenesis

Key Points:

• Highlights the advantages of conditional (Cre/lox-based) gene deletion:

  • Avoids embryonic lethality.

  • Enables analysis of gene function in adults.

  • Dissects gene function in specific organs or systems.

Explanation of Visuals:

• Text slide listing key advantages in a clear format.

Glossary:

• Mutagenesis: Process of inducing mutations (e.g., deletions or modifications).

Key Takeaway:
Conditional mutagenesis provides spatiotemporal control over gene inactivation, avoiding systemic developmental defects.

Slide 64: Placing a Stop = Blocking a Bridge

Key Points:

• A STOP cassette is placed between loxP sites upstream of a gene.

• Without Cre: transcription is blocked (bridge is "closed").

• With Cre: the STOP cassette is removed, allowing gene expression ("bridge opened").

Explanation of Visuals:

• Top: Gene blocked by a STOP cassette.

• Bottom: Gene is reactivated after Cre-mediated deletion of the cassette.

Glossary:

• STOP cassette: A DNA sequence that prevents transcription until removed by Cre recombinase.

Key Takeaway:
The STOP strategy allows Cre-dependent gene activation, enabling temporal or spatial control of gene expression.

Slide 65: Placing a Tag = Using a Reporter (Cre Reporter Lines)

Key Points:

• Reporter systems like LacZ or GFP are used to visualize Cre activity.

• After Cre recombination:

  • Reporter gene is activated.

  • Fluorescence (e.g., red signal) shows where Cre was expressed.

• Used for lineage tracing or confirming tissue targeting.

Explanation of Visuals:

• Diagrams of recombination event.

• Microscopy image shows red-labeled cells in Cre+ tissues.

Glossary:

• Reporter gene: A gene whose product is easily detectable (e.g., GFP, LacZ).

• Lineage tracing: Tracking cell fate using genetic markers.

Key Takeaway:
Reporter tags help visualize Cre activity and confirm where and when gene activation or deletion occurred.

Slide 66: Conditional Targeting – Inducible Cell Type-Specific Mutagenesis

Key Points:

• This system allows controlled gene deletion in specific cell types at a chosen time.

• It uses a modified Cre enzyme called CreERᵀ², which is inactive until tamoxifen is given.

• Tamoxifen, a synthetic hormone, binds to CreERᵀ² and enables it to enter the nucleus.

• Once inside the nucleus, CreERᵀ² can recombine DNA at loxP sites, leading to gene deletion.

• The system allows for temporal (time-specific) and spatial (tissue-specific) gene knockout.

Explanation of Visuals:

• Left side: The mouse cell nucleus shows CreERᵀ² bound to tamoxifen → enters nucleus → recombination at loxP sites occurs → gene deletion.

• Right side: Illustration of a mouse being injected with tamoxifen.

  • Before injection: CreERᵀ² is present but inactive.

  • After injection: Tamoxifen activates CreERᵀ² → recombination happens in the mouse's cells.

Glossary:

• CreERᵀ²: A fusion of Cre recombinase with a mutated estrogen receptor that only becomes active in the presence of tamoxifen.

• Tamoxifen: A drug that activates CreERᵀ² by enabling its movement into the nucleus.

• loxP: A short DNA sequence where Cre acts to cut or recombine DNA.

• Inducible gene deletion: A system that deletes a gene only after activation (e.g., via tamoxifen).

Key Takeaway:
This inducible Cre-loxP system enables precise gene deletion in specific tissues and at selected times by administering tamoxifen — making it a powerful tool for studying gene function in vivo.

Slide 67: Photoactivatable Cre

Key Points:

• Cre recombinase is split into two inactive fragments (CreN59 and CreC60).

• These fragments are fused to light-sensitive proteins (nMag and pMag).

• When exposed to blue light (BL), the proteins dimerize (bind together), forming an active Cre enzyme.

• The active Cre mediates site-specific recombination between two loxP sites.

• This system allows precise temporal control: recombination occurs only when light is applied.

Explanation of Visuals:

• On the left, inactive Cre fragments (gray) are shown.

• Blue light induces dimerization of nMag/pMag (blue/pink), activating Cre (purple).

• On the right, activated Cre recombines the DNA between loxP sites, excising the segment.

Glossary:

• Cre recombinase: An enzyme that recombines DNA at specific loxP sites.

• loxP: A short DNA sequence recognized by Cre, used in gene editing.

• Photoactivatable: Can be switched on using light.

• Dimerization: The joining of two protein fragments to form a functional unit.

Key Takeaway:
Photoactivatable Cre systems allow researchers to control gene recombination in specific tissues at specific times using light — adding precision to genetic studies.

Slide 68: First Inducible Knockout (KO)

Key Points:

• This slide highlights the first inducible knockout system, developed by Schwenk et al., 1998.

• The system enables temporal and spatial control of gene deletion.

• Cre recombinase expression is placed under the control of a regulatable promoter (e.g., inducible via drugs or tissue-specific promoters).

• When Cre is expressed, it mediates recombination at loxP sites, deleting the target gene only under specific conditions.

Explanation of Visuals:

• Left diagram (A) shows the genetic construct: the Cre gene is controlled by regulatory sequences, enabling its inducible expression.

• Right panel (B) shows flow cytometry data:

  • Wild-type (wt) shows normal IgM/IgD expression.

  • After induction with Cre, IgM and IgD expression is altered, indicating successful knockout of the target gene in specific B cells.

Glossary:

• Inducible KO: A knockout system where gene deletion is activated by a signal (e.g., drug or time point).

• Flow cytometry: A technique to measure protein expression on individual cells.

• IgM/IgD: Immunoglobulin markers on B cells.

• Promoter: DNA region that controls when and where a gene is turned on.

Key Takeaway:
This was the first demonstration of a system where Cre-mediated gene knockout could be precisely timed or targeted to specific tissues — a foundational tool for conditional genetics.

Slide 69: Pharmacologically Inducible Cre

Key Points:

• A modified Cre recombinase (CreER or CreERT2) can be activated by a drug, typically tamoxifen.

• Without tamoxifen:

  • CreERT remains in the cytoplasm.

• After tamoxifen binds:

  • CreERT translocates to the nucleus.

  • Recombination between loxP sites occurs.

• Offers temporal control over gene deletion.

Explanation of Visuals:

• Diagram showing:

  • Cytoplasmic retention of CreERT.

  • Activation and nuclear import after drug exposure.

  • Subsequent loxP recombination.

Glossary:

• CreERT: Cre fused with a modified estrogen receptor.

• Tamoxifen: A drug used to induce CreERT nuclear activity.

Key Takeaway:
Pharmacologically inducible Cre systems enable controlled gene modification in response to drug administration.

Slide 70: The Cuts Are Repaired – With Mistakes

Key Points:

• This slide explains how cells repair double-strand DNA breaks (DSBs) — a key concept in gene editing.

• When a designer nuclease (e.g., CRISPR-Cas9) cuts DNA, the cell tries to repair the break.

• Two outcomes are shown:

  • Non-homologous end joining (NHEJ): Fast but error-prone → often results in insertions or deletions (indels).

  • These indels can disrupt genes and are used to knock out gene function.

Explanation of Visuals:

• Diagram shows a DNA double-strand break followed by:

  • Cutting by nuclease

  • Rejoining by cellular repair enzymes

  • Resulting mutations (indels) which can alter gene function

Glossary:

• DSB (Double-strand break): A complete break across both strands of DNA.

• NHEJ (Non-homologous end joining): A quick but inaccurate DNA repair method that can introduce mutations.

• Indel: Insertion or deletion of bases during DNA repair.

• Gene knockout: Disruption of gene function through mutation.

Key Takeaway:
When designer nucleases cut DNA, the cell repairs the damage — often introducing mutations. This natural error-prone process is used to disable genes in gene-editing experiments.

Slide 71: The First Designer Nucleases

Key Points:

• This slide introduces engineered nucleases that allow scientists to cut DNA at precise sites.

• Key systems shown:

  • Zinc Finger Nucleases (ZFNs) – introduced ~2008

  • TALENs (Transcription Activator-Like Effector Nucleases) – introduced ~2011

• These tools consist of a DNA-binding domain (that recognizes a specific sequence) and a nuclease domain (that cuts DNA).

• Important feature: Scientists can choose where the cut happens, allowing targeted gene modification.

Explanation of Visuals:

• The slide shows schematic representations of ZFNs and TALENs binding to DNA and making cuts at desired sites.

• Emphasized phrase: “We can decide where they cut” — highlighting the precision of designer nucleases.

Glossary:

• Zinc Finger Nucleases (ZFNs): Engineered proteins that bind DNA via zinc finger domains and cut it using FokI nuclease.

• TALENs: Proteins that use transcription activator-like domains to bind DNA and FokI to cleave it.

• Designer nuclease: A customizable enzyme that cuts DNA at specific user-defined sequences.

Key Takeaway:
ZFNs and TALENs were the first technologies to enable scientists to cut DNA at precise locations, allowing targeted gene editing.

Slide 72: Cas9 – Simple to Program Endonuclease

Key Points:

• This slide introduces CRISPR-Cas9, a powerful and easy-to-program gene-editing system.

• Cas9 uses a guide RNA (gRNA) to direct it to a specific DNA sequence.

• Unlike ZFNs or TALENs, Cas9 does not need custom protein engineering — only a new guide RNA is required for each target.

• The system is composed of:

  • Cas9 protein (endonuclease)

  • gRNA (or crRNA + tracrRNA in the native system)

Explanation of Visuals:

• Diagram shows Cas9 bound to DNA, directed by guide RNA to make a site-specific double-strand cut.

Glossary:

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): A bacterial immune system adapted for gene editing.

• Cas9: An enzyme that cuts DNA at a location specified by the guide RNA.

• Guide RNA (gRNA): A synthetic RNA that directs Cas9 to the target sequence.

• crRNA / tracrRNA: Components of the natural bacterial guide system, often fused into a single gRNA in labs.

• Endonuclease: An enzyme that cuts DNA inside a sequence (not at the end).

Key Takeaway:
CRISPR-Cas9 revolutionized gene editing by making it fast, cheap, and easily programmable, using simple guide RNAs to direct DNA cutting with high precision.

Slide 73: Gene Editing by CRISPR/Cas

Key Points:

• Introduces CRISPR/Cas technology for genome editing.

• Developed by Emmanuelle Charpentier and Jennifer Doudna (Nobel Prize 2020).

• CRISPR/Cas allows scientists to cut and modify DNA precisely and efficiently.

Explanation of Visuals:

• Top: Photo of the inventors.

• Bottom: Symbolic CRISPR scissors and DNA.

Glossary:

• CRISPR/Cas: A bacterial immune system adapted for genome editing in other organisms.

Key Takeaway:
CRISPR/Cas is a powerful, programmable system for gene editing that revolutionized molecular biology.

Slide 74: Choice of gRNAs and Use of RNA

Key Points:

• CRISPR works through a guide RNA (gRNA) that directs Cas9 to a specific DNA sequence.

• gRNA binds to the complementary DNA.

• Cas9 makes a double-strand break at the target site.

Explanation of Visuals:

• Diagrams showing:

  • gRNA-Cas9 complex binding to target DNA.

  • DNA cleavage between PAM site and gRNA binding site.

Glossary:

• gRNA (guide RNA): RNA molecule that guides Cas9 to the DNA target.

• PAM (Protospacer Adjacent Motif): A short DNA sequence required for Cas9 activity.

Key Takeaway:
CRISPR/Cas specificity is determined by the gRNA, which enables programmable DNA targeting.

Slide 75: How to Edit a Targeted Gene

Key Points:

• CRISPR-induced DNA breaks can be repaired via:

  • NHEJ (non-homologous end joining) → may introduce small insertions/deletions.

  • HDR (homology-directed repair) → allows precise edits using a donor template.

• Method depends on desired outcome (disruption vs. correction).

Explanation of Visuals:

• Diagrams show NHEJ creating indels and HDR enabling sequence replacement.

Glossary:

• NHEJ: Fast, error-prone repair mechanism.

• HDR: High-fidelity repair pathway using a homologous DNA template.

Key Takeaway:
CRISPR can be used to disrupt genes or precisely edit them, depending on the DNA repair pathway engaged.

Slide 76: Electroporation of gRNAs

Key Points:

• gRNAs and Cas9 protein can be introduced into embryos using electroporation.

• Electric pulses open cell membranes, allowing large molecules like CRISPR components to enter.

• Fast and efficient method for in vivo gene editing.

Explanation of Visuals:

• Diagram shows microinjection and electroporation process.

Glossary:

• Electroporation: A method that uses electricity to introduce substances into cells.

Key Takeaway:
Electroporation enables efficient delivery of CRISPR reagents into embryos without using viral vectors.

Slide 77: First CRISPR/Cas9 Genome Modification

Key Points:

• Demonstration of successful gene editing using CRISPR/Cas9.

• Gels and sequencing show mutation introduction and confirmation of editing.

• Validates CRISPR as a functional genome-editing tool in mammalian cells.

Explanation of Visuals:

• Top: Gel electrophoresis of edited vs. control DNA.

• Bottom: DNA sequence traces confirming indels.

Glossary:

• Genome modification: Permanent change in an organism’s DNA.

• Indel: Insertion or deletion mutation.

Key Takeaway:
CRISPR/Cas9 was successfully used to introduce targeted mutations in mammalian cells.

Slide 78: CRISPR-Induced Sequences

Key Points:

• Shows sequence data from wild-type and CRISPR-edited DNA.

• Edited sequences have small insertions or deletions caused by NHEJ.

• Confirms the site-specific activity of Cas9 guided by gRNA.

Explanation of Visuals:

• Sanger sequencing traces showing mismatches/frameshifts at the expected cut site.

Glossary:

• Sanger sequencing: A method to determine the nucleotide sequence of DNA.

Key Takeaway:
CRISPR editing can be confirmed through sequencing by identifying characteristic indels near the target site.

Slide 79: Cloning and Gene Editing – CRISPR in Non-Human Primates

Key Points:

• Describes the use of CRISPR for editing genes in monkey embryos.

• Cas9 and gRNA are injected into zygotes.

• Produces genetically modified non-human primates (NHPs) for biomedical research.

Explanation of Visuals:

• Diagram showing CRISPR injection into fertilized eggs and development of genetically altered offspring.

Glossary:

• Non-human primates (NHPs): Monkeys used as models for studying human disease.

Key Takeaway:
CRISPR is powerful enough to be applied in primate embryos, advancing disease modeling and neuroscience.

Slide 80: Mutations in Twin Monkeys (Qin et al., 2015)

Key Points:

• Study shows CRISPR/Cas used to generate gene-edited twin monkeys.

• Mutations were successfully introduced and inherited.

• Demonstrates feasibility of CRISPR in higher mammals.

Explanation of Visuals:

• Photo of the twin monkeys born from gene-edited embryos.

Glossary:

• Germline editing: Genetic changes introduced into embryos that are heritable.

Key Takeaway:
CRISPR can be used to create genetically modified primates, marking a major leap in translational genetics.

Slide 81: Cloned Monkeys

Key Points:

• Presentation of cloned primates generated via somatic cell nuclear transfer (SCNT).

• Demonstrates the cloning of genetically identical monkeys from the same nuclear DNA.

• Used for studying gene function and modeling human diseases.

Explanation of Visuals:

• Image of early embryonic stage and a photograph of the cloned monkeys.

• Diagram shows nucleus transfer from a donor cell into an enucleated egg.

Glossary:

• SCNT (Somatic Cell Nuclear Transfer): Transferring the nucleus of a somatic cell into an egg cell to create a clone.

Key Takeaway:
Cloning in primates has been achieved, allowing the creation of identical animals for controlled genetic studies.

Slide 82: Oocyte Microinjection

Key Points:

• This slide illustrates how CRISPR-Cas9 components are directly injected into fertilized mouse oocytes (zygotes).

• The goal is to perform targeted gene editing at the earliest embryonic stage.

• Components injected:

  • Cas9 protein

  • Guide RNA (gRNA)

  • Optional: a donor DNA template for targeted insertion or replacement

• The injection targets the pronucleus of the fertilized oocyte.

• Two outcomes are possible:

  • Target cleavage: Cas9 induces a double-strand break (DSB).

  • Target insertion: If a donor template is present, precise modification via homologous recombination can occur.

Explanation of Visuals:

• Top: Shows Cas9/gRNA being injected into the oocyte.

• Bottom: Zoom-in view of the nucleus showing:

  • Left: Target gene being cut by Cas9.

  • Right: Option for inserting a new DNA sequence using a donor template.

Glossary:

• Oocyte: An unfertilized egg cell.

• Pronucleus: The nucleus of a sperm or egg before they fuse — the preferred site for genetic injection.

• Microinjection: A precise technique to introduce molecules into cells using a fine needle.

• Donor template: A piece of DNA used to guide repair by homology, enabling targeted sequence insertion.

• Knock-in: A targeted insertion of new genetic material.

Key Takeaway:
CRISPR-Cas9 allows gene editing at the zygote stage by microinjecting Cas9, gRNA, and optional donor DNA directly into the oocyte, enabling fast generation of genetically modified animals.

Slide 83: Precise Modification by CRISPR/Cas9

Key Points:

• This slide shows the molecular details of precise gene editing using CRISPR/Cas9 with a donor template.

• Key steps:

  1. Cas9 + gRNA bind to the target DNA sequence.

  2. Cas9 makes a double-strand break (DSB).

  3. If a homologous donor DNA is provided, the cell can use it to repair the break accurately via homology-directed repair (HDR).

  4. This allows for precise insertion or replacement of DNA at the cut site.

Explanation of Visuals:

• Shows Cas9 + gRNA binding and cutting the target site.

• Highlights the use of a donor DNA with homology arms to guide the repair.

• Visual outcome: precise editing of the target gene.

Glossary:

• Homology-directed repair (HDR): A high-fidelity repair pathway that uses a homologous DNA template to accurately fix a break.

• Spacer and scaffold: Parts of the guide RNA — the spacer matches the target, and the scaffold interacts with Cas9.

• Precise edit / knock-in: A deliberate insertion or correction of DNA at a specific genomic site.

Key Takeaway:
CRISPR-Cas9 can be used for precise genome editing when paired with a donor DNA template, enabling researchers to insert, replace, or correct genes at defined locations via HDR.

Slide 84: Tissue-Specific Editing by CRISPR/Cas

Key Points:

• Describes tissue-specific gene editing using Cas9 under a cell-specific promoter.

• Only tissues expressing the promoter activate CRISPR editing.

• Example: Cas9 in liver cells only → edits occur only in liver.

Explanation of Visuals:

• Bar graph shows gene expression differences across tissues post-editing.

• Diagram of tissue-specific promoter driving Cas9 expression.

Glossary:

• Promoter: DNA sequence that controls gene transcription.

• Tissue-specific promoter: Active only in certain cell types.

Key Takeaway:
Tissue-targeted gene editing ensures mutations occur only in the desired cell type, improving specificity.

Slide 85: Z-DNA Binding Domain

Key Points:

• Z-DNA is a left-handed form of DNA, associated with genomic instability and stress response.

• Z-DNA-binding proteins can be studied using genome editing tools.

• Implicated in transcriptional regulation and immune activation.

Explanation of Visuals:

• Cartoon showing Z-DNA and proteins binding specifically to this unusual DNA structure.

Glossary:

• Z-DNA: An alternative DNA conformation with left-handed helical structure.

• DNA binding domain: A part of a protein that interacts with specific DNA shapes or sequences.

Key Takeaway:
Studying proteins that bind Z-DNA can help understand regulatory and stress-response mechanisms.

Slide 86: Precise Modification by CRISPR/Cas (Protein Structure Focus)

Key Points:

• CRISPR can be used to modulate protein structure or study mutations that affect protein folding.

• Useful in functional protein studies, structural biology, and disease modeling.

Explanation of Visuals:

• Visual of protein domains and structural elements.

• Illustration of how mutations can impact conformation.

Glossary:

• Protein folding: Process by which a protein assumes its 3D shape.

• Domain: Functionally distinct region of a protein.

Key Takeaway:
CRISPR allows researchers to manipulate protein domains and study structural-functional relationships.

Slide 87: Cas9 Knockout vs. Knock-in

Key Points:

• Comparison of gene knockout vs. knock-in using CRISPR:

  • Knock-out: via NHEJ, causes gene disruption.

  • Knock-in: via HDR, inserts new sequence (e.g., tags or corrects mutations).

• Both approaches use Cas9 to cut the DNA.

Explanation of Visuals:

• Top: Cas9 induces indels (knockout).

• Bottom: Homology template used for knock-in.

• Clear schematic comparison.

Glossary:

• Knockout: Gene inactivation.

• Knock-in: Inserting a new or corrected gene sequence.

Key Takeaway:
CRISPR enables both gene inactivation and precise modification depending on the repair pathway used.

Slide 88: Base Editing by CRISPR/Cas

Key Points:

• This slide introduces a refined CRISPR-Cas9 technique called base editing, which allows precise single-base changes in DNA without creating double-strand breaks (DSBs).

• Base editing uses a modified Cas9 enzyme (either a nickase or dead Cas9, dCas9) that is fused to a cytidine deaminase enzyme.

• The complex binds the DNA at a specific target site guided by gRNA, and instead of cutting, it chemically alters a base.

• Specifically, cytidine (C) is deaminated to uracil (U), which is then recognized as thymine (T) during DNA replication or repair.

• A base excision repair inhibitor is also included in the complex to prevent the cell from undoing the edit.

• If the modified strand is used as a template, the change is permanently incorporated via mismatch repair.

Explanation of Visuals:

• Left side: Shows Cas9 fused to a cytidine deaminase. The Cas9 binds DNA via the gRNA scaffold and spacer.

• Middle: Illustrates binding to the DNA target and PAM site without introducing a DSB.

• Right side: Shows C-to-U conversion and how mismatch repair can preserve the edit, resulting in a C→T change in the genome.

Glossary:

• Base editing: A genome editing method that directly converts one base pair into another without cutting both DNA strands.

• Cas9 nickase (nCas9): A Cas9 variant that cuts only one DNA strand.

• dCas9 (dead Cas9): A mutated Cas9 that binds DNA but does not cut it.

• Cytidine deaminase: An enzyme that converts cytidine (C) into uracil (U).

• Mismatch repair: A DNA repair mechanism that fixes base-pair mismatches — here, it helps incorporate the edited base.

• PAM (Protospacer Adjacent Motif): A short DNA sequence required for Cas9 to bind and act at the target site.

• Scaffold/spacer: Parts of the guide RNA; the spacer targets the DNA, the scaffold binds Cas9.

Key Takeaway:
Base editing with CRISPR/Cas9 allows precise single-base conversions (e.g., C→T) without cutting DNA, offering a safer and more controlled alternative to traditional gene editing approaches.

Slide 89: Activation/Repression by CRISPR/Cas

Key Points:

• CRISPR/Cas can be modified for gene regulation rather than cutting DNA.

• dCas9 (dead Cas9) binds DNA without cutting it.

• Fused to:

  • Transcriptional activators → increase gene expression.

  • Repressors → decrease gene expression.

Explanation of Visuals:

• Diagram shows dCas9 fused to an activator domain targeting a gene promoter.

Glossary:

• dCas9: A catalytically inactive Cas9 used for gene regulation instead of cutting.

• Transcriptional regulation: Controlling how much of a gene is expressed.

Key Takeaway:
CRISPR/dCas9 enables gene expression to be turned up or down without altering the DNA sequence.

Slide 90: Epigenetics by CRISPR/Cas

Key Points:

• CRISPR/dCas9 can also modify epigenetic marks, such as:

  • DNA methylation

  • Histone acetylation

• Achieved by fusing dCas9 to epigenetic modifiers.

Explanation of Visuals:

• Illustrates CRISPR/dCas9 delivering epigenetic changes to target loci.

Glossary:

• Epigenetics: Heritable changes in gene function without changes to the DNA sequence.

• Methylation: A chemical tag that typically silences gene expression.

Key Takeaway:
CRISPR tools can now modulate gene activity through epigenetic mechanisms, expanding control beyond sequence editing.

Slide 91: Mitochondrial Genome Modification by Cas9

Key Points:

• Challenges exist in delivering CRISPR/Cas9 to mitochondria.

• Alternative strategies involve:

  • Mitochondria-targeted nucleases (e.g., mitoTALENs, ZFNs).

• Aim: Treat mitochondrial diseases by correcting mtDNA mutations.

Explanation of Visuals:

• Microscopy images of mitochondria and fluorescent markers.

• Illustration of mtDNA modification challenges.

Glossary:

• mtDNA: Mitochondrial DNA.

• MitoTALEN: A TALEN modified to target mitochondrial DNA.

Key Takeaway:
Gene editing in mitochondria is still under development but holds potential for treating mitochondrial diseases.

Slide 92: Typing and Documentation

Key Points:

• Emphasizes importance of systematic typing and record-keeping in genetically modified animal research.

• Ensures:

  • Correct genotype identification.

  • Reproducibility.

  • Regulatory compliance.

Explanation of Visuals:

• Title slide only, introducing the topic.

Glossary:

• Typing: Determining an organism’s genetic makeup.

• Documentation: Recording and managing genetic data.

Key Takeaway:
Accurate genetic typing and documentation are critical for managing genetically engineered animal colonies.

Slide 93: Typing Strategy – Overview

Key Points:

• Slide 93: Transgene Nomenclature

  Key Points:

  • The standard format for naming transgenic mice is explained using an example:

    • Tg(HBB-GH1)3King

  • Components:

    • Tg: Indicates a transgene.

    • (HBB-GH1): Specifies the transgene or gene construct (e.g., human growth hormone under the hemoglobin beta promoter).

    • 3: Founder line number.

    • King: Lab or researcher code (person or lab of origin).

  Glossary:

  • Transgene: A foreign gene inserted into an organism.

  • Promoter: A DNA sequence that drives gene expression.

  • Founder: The original genetically modified animal used to establish a transgenic line.

  Key Takeaway:
  Transgene nomenclature clearly encodes the gene construct, promoter, founder line number, and lab origin.

  ---

  Slide 94: Targeted Mutation Nomenclature

  Key Points:

  • Format for targeted mutations (gene knockouts, knock-ins) is shown:

    • Ldlr^tm1Her

  • Components:

    • Ldlr: Gene symbol (e.g., low-density lipoprotein receptor).

    • tm1: Targeted mutation 1.

    • Her: Lab code (Joachim Herz, in this case).

  Glossary:

  • Targeted mutation (tm): A gene that has been precisely altered (e.g., via homologous recombination or CRISPR).

  • Allele type: Classification of the mutation (e.g., tm1 = first targeted mutation).

  • Lab code: Refers to the scientist or lab where the mouse line originated.

  Key Takeaway:
  Targeted mutation nomenclature identifies the gene, mutation type, and origin of the modification.

  ---

  Slide 95: Targeted Mutation Nomenclature (Background)

  Key Points:

  • Shows how genetic background is included:

    • 129S7/SvEvBrd-Ldlr^tm1Her/J

  • Components:

    • 129S7/SvEvBrd: Refers to the mouse strain used (ES cell line background).

    • Ldlr^tm1Her: The targeted allele.

    • /J: Lab or source (e.g., Jackson Laboratory).

  Glossary:

  • 129 background: A strain commonly used for ES cell derivation in gene targeting.

  Key Takeaway:
  The full name of a mutant line includes both the genetic modification and the strain background.

  ---

  Slide 96: Targeted Mutation Nomenclature (Backcrossed Line)

  Key Points:

  • Shows a congenic line:

    • C57BL/6.129S7/SvEvBrd-Ldlr^tm1Her/J

  • Components:

    • C57BL/6: Indicates that the mouse has been backcrossed to the B6 background.

    • The rest refers to the original 129-derived allele and lab code.

  Glossary:

  • Congenic strain: A mouse line bred onto a defined genetic background (e.g., B6) for many generations.

  Key Takeaway:
  Nomenclature tracks both the modification and the backcrossing history of genetically engineered mouse lines.

  ---

  Slide 97: Allele Types

  Key Points:

  • Lists different allele type suffixes and what they mean:

    • tm = targeted mutation

    • tmn = targeted knock-in

    • Gt = gene trap

    • em = endonuclease-mediated (e.g., CRISPR)

    • allele with name = ENU-induced or spontaneous mutation

  Glossary:

  • Knock-in: Insertion of a specific DNA sequence into a target locus.

  • Gene trap: A method using random insertion to disrupt gene function.

  • ENU mutation: Random mutation induced by ethylnitrosourea, a chemical mutagen.

  • CRISPR (em): Endonuclease-mediated, used for modern precision genome editing.

  Key Takeaway:
  Allele names reflect the method of generation and are standardized for clarity in research and publication.

  ---

  Slide 98: Documentation of Typing Results

  Key Points:

  • Shows how genotypes are recorded:

    • For autosomal genes: mut/mut, mut/wt, or wt/wt

    • For X- and Y-linked genes: use xmut/xmut, xmut/xwt, etc.

    • Separate conventions for males vs. females in X/Y-linked cases.

  Glossary:

  • Genotyping: Determining the genetic makeup (alleles) of an animal.

  • Hemizygous: Having only one copy of a gene (e.g., X-linked genes in males).

  • mut/wt: One mutant allele, one wild-type allele (heterozygous).

  • xwt/xmut: Format used to indicate X-linked genotypes.

  Key Takeaway:
  Standardized notation is used for reporting mouse genotypes, especially when tracking both alleles and sex-linked mutations.

Slide 99: Documentation of Typing Results

Key Points:

• Typing results must be well-documented and traceable.

• Essential elements:

  • Animal ID

  • PCR result

  • Genotype

  • Date and analyst initials

• Centralized records improve reproducibility and breeding management.

Explanation of Visuals:

• Table format of standardized genotype documentation fields.

Glossary:

• PCR (Polymerase Chain Reaction): A common genotyping method that amplifies specific DNA sequences.

Key Takeaway:
Systematic documentation of genotyping data supports transparency, reproducibility, and compliance in research.

Slide 101: Zebrafish Transgenesis

Key Points:

• Zebrafish are widely used for in vivo gene function studies.

• Transgenesis typically involves:

  • Tol2 transposon system to integrate DNA.

  • Injection into fertilized eggs.

• Allows real-time imaging of development using fluorescent reporters.

Explanation of Visuals:

• Diagram of injection method and example of GFP-expressing zebrafish.

Glossary:

• Tol2 transposon: A mobile DNA element used to insert transgenes into genomes.

• GFP (Green Fluorescent Protein): A fluorescent tag used to visualize gene expression.

Key Takeaway:
Zebrafish are powerful tools for studying gene function, particularly with visible fluorescent markers.

Slide 102: Organisms That Can Be Transgenized

Key Points:

• Many model organisms can be genetically modified, including:

  • Mice, rats, zebrafish, flies, worms, and even primates

• Each species has specific tools and protocols for transgenesis.

Explanation of Visuals:

• Colorful grid of organisms commonly used in genetic research.

Glossary:

• Transgenesis: The process of introducing foreign DNA into an organism’s genome.

Key Takeaway:
Transgenesis is applicable across many model species, enabling broad exploration of gene function.