Biotechnology 2 Lecture Notes

Exactly 100 vocabulary flashcards covering biotechnology techniques including CRISPR/Cas9, iPSCs, RNAi, RT-PCR, and gene expression analysis based on the provided lecture notes.

Transgene

A gene that has been created through cloning and introduced into cells.

PCR and Cloning Application

Techniques that allow researchers to amplify any gene and place it under the control of any promoter.

Recombinant DNA Delivery

Process where recombinant DNA is placed into a retrovirus for integration into eukaryotic cells.

Transduction

The process of using a viral vector to deliver recombinant DNA into cells.

Stem Cell

A relatively unspecialized cell that can reproduce itself indefinitely and be induced to differentiate into specialized cell types.

Pluripotent

The capability of a stem cell to differentiate into many different cell types.

Yamanaka Factors

A set of $4$ key transcription factors that can reprogram a differentiated cell into a stem cell.

Fibroblast

A type of differentiated cell used in Yamanaka’s research to generate stem cells via retroviral transduction.

Induced Pluripotent Stem Cells (iPSCs)

Stem cells generated by the retroviral transduction of key transcription factors into a differentiated cell.

iPSC Pluripotency Evidence

The ability of iPSCs to form a variety of cell types when injected into mouse skin.

Skin Biopsy

A procedure used to obtain cells from a patient to create patient-specific iPS cells.

Gene Targeting in iPSCs

Technique used to repair disease-causing mutations in patient-specific induced pluripotent stem cells.

In Vitro Differentiation

The process of growing repaired or patient-specific iPS cells into affected cell types for drug screening or transplantation.

Oct4

One of the four transcriptional factors used to generate induced pluripotent stem cells.

Sox2

A transcription factor included in the Yamanaka factors for cellular reprogramming.

Klf4

A key transcription factor identified by Yamanaka for the creation of iPSCs.

c-Myc

A transcription factor used alongside Oct4, Sox2, and Klf4 to reprogram fibroblasts.

RNA-sequencing (RNA-seq)

A technique that provides a snapshot of which protein-coding genes are being transcribed in a cell at a specific time.

Reverse-transcription Polymerase Chain Reaction (RT-PCR)

A technique used to track the relative amounts of specific mRNAs over time by amplifying cDNA.

cDNA

Complementary DNA synthesized from mRNA by reverse transcriptase, used as a proxy for mRNA abundance.

Reverse Transcriptase

An enzyme used in RT-PCR to convert extracted mRNA into cDNA.

Gel Electrophoresis in RT-PCR

A method used to separate PCR products where band intensity signifies the amount of the original cDNA.

Tissue Lysis

The process of breaking up a specific cell or tissue to isolate mRNAs for RNA sequencing.

Cellular Context Loss

A disadvantage of RNA-seq where breaking up tissue prevents knowing which specific cells expressed an mRNA.

In-situ Hybridization

A technique that allows the detection of gene expression within intact tissues or organisms using fluorescently labeled probes.

DNA Probe

A short, fluorescently labeled oligonucleotide designed to base-pair with a target mRNA in native tissue.

Oligonucleotides

Short sequences of DNA, similar to primers, used as probes in hybridization experiments.

Fluorophore

A fluorescent chemical compound used to label DNA probes or antibodies for visualization.

Multiplexing in Hybridization

The simultaneous use of multiple probes conjugated to different fluorophores to detect different mRNA targets at once.

Drosophila Embryo Projection

An example where in-situ hybridization visualizes $5$ different mRNAs simultaneously in an intact organism.

BRCA1 Coding Strand Sequence

$$5' ATG GAT TTA TCT GCT 3'$$

Immunostaining

A technique using antibodies to detect specific protein targets in intact cells or tissues.

Antigen

The specific target of interest, typically a protein, that is recognized by a primary antibody.

Primary Antibody

An antibody that specifically recognizes and binds to a target protein of interest.

Secondary Antibody

An antibody conjugated to a fluorophore that specifically binds to a primary antibody.

Signal Amplification in Immunostaining

Occurs when multiple secondary antibodies bind to a single primary antibody, increasing the fluorescent signal.

Multiplexing in Immunostaining

Using multiple primary and secondary antibodies with different fluorophores to look at multiple protein targets simultaneously.

Vasa Protein

A protein targeted by antibodies to identify primordial germ cells in Drosophila embryos.

Cyan Dye in Embryo Images

A fluorescent dye used in images to specifically bind to and visualize DNA.

Endoderm-expressed Protein

A target for immunostaining used alongside Vasa to distinguish different cell types in the embryo.

Classical Genetics

An approach starting with an interesting phenotype to identify the underlying genotype or mutated gene.

Mutagen

A substance used in classical genetic screens to increase the incidence of DNA mutations and phenotypic variety.

Unbiased Genetic Screen

A screen where new genes never previously associated with a phenotype can be discovered without prior assumptions.

Reverse Genetics

An approach that starts with a specific gene of interest and assesses its function by removing it and examining the resulting phenotype.

RNA Interference (RNAi)

A reverse genetics technique that uses siRNA to deplete target mRNAs and silence specific genes.

Small Interfering RNAs (siRNAs)

Molecules that form complexes with proteins to degrade complementary mRNA post-transcriptionally.

C. elegans in RNAi

A free-living nematode used in experiments to study gene knockdown by feeding it bacteria expressing double-stranded RNA.

E. coli in RNAi experiments

Bacteria engineered to express double-stranded RNA which is then processed into siRNA within the nematode host.

Nuclear Migration Defects

Phenotypic result observed in C. elegans embryogenesis following the knockdown of specific mRNAs via RNAi.

CRISPR/Cas9

A gene-editing system adapted from a bacterial defense mechanism that allows for the creation or correction of mutations.

CRISPR Loci

Regions in bacterial genomes where foreign viral DNA is integrated between repeat host DNA sequences.

Protospacers

Foreign DNA sequences integrated into the bacterial CRISPR locus to provide immunity against future infections.

pre-crRNA

The initial transcript of a CRISPR locus that is later processed into individual crRNAs.

crRNA (CRISPR RNA)

Processed RNA containing foreign DNA sequences that bind to Cas proteins to guide DNA cleavage.

Cas Protein

A nuclease that mediates the cleavage of foreign DNA when guided by a complementary crRNA sequence.

RNA Directed DNA Cleavage

The mechanism by which the crRNA-Cas complex recognizes and cuts viral DNA through base pairing.

Protospacer Adjacent Motif (PAM)

A specific sequence of nucleotides required for CRISPR/Cas cleavage that is present in viral DNA but not the bacterial genome.

Cas9 PAM Sequence

$$5' NGG 3'$$

N in PAM

A notation indicating that any of the four nucleotides can occupy that position in the motif.

Nuclease

An enzyme, such as Cas9, capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids.

Charpentier and Doudna

Researchers who worked out the molecular mechanisms of CRISPR/Cas as a bacterial immunity system in $2012$.

gRNA (guide RNA)

A simplified single RNA component that replaces the crRNA and secondary RNA to target Cas9 to specific DNA sequences.

sgRNA

An alternative name for single guide RNA used in CRISPR/Cas9 systems.

Cas9 Utility

A protein favored for gene editing because it requires no protein cofactors for cleavage and works across many organisms.

Off-target Cutting

A caveat of CRISPR/Cas9 where the enzyme cuts at multiple sites with sequence homology to the target.

Non-homologous End Joining (NHEJ)

An error-prone DNA repair process that typically leads to random additions or deletions of nucleotides.

Frameshift Mutation via CRISPR

Created by targeting a double-stranded break to an early exon, leading to a non-functional protein after NHEJ repair.

Homology Directed Repair (HDR)

A precise DNA repair pathway that uses a user-defined template to incorporate specific edits into the genome.

Repair Template

A single or double-stranded DNA molecule with sequence homology used during HDR to fix mutations or insert DNA.

FLAG Protein Tag

A specific peptide sequence inserted into an exon to facilitate protein purification or immunostaining.

dCas9 (Dead Cas9)

A modified Cas9 that can target a specific sequence but lacks the ability to cut DNA.

Base Editing

A modification of CRISPR using dCas9 fused to a deaminase enzyme to convert one nucleotide to another without double-stranded breaks.

Deaminase

An enzyme used in base editing to change nucleotides on DNA strands during the repair process.

Somatic Cell Therapy

CRISPR treatments targeted at non-reproductive cells, ensuring edits are not passed to offspring.

Sickle Cell Disease Correction

An FDA-approved therapy involving the CRISPR-editing of a patient's blood stem cells.

CCR5 Gene

The gene targeted for disabling in a controversial $2018$ study to prevent HIV transmission in human embryos.

Pre-crRNA Processing

The step where a long CRISPR transcript is cut into individual crRNAs bound to Cas proteins.

RNA-guided Targeting

A process similar in siRNA and CRISPR where an RNA molecule directs a protein complex to a specific target.

Multiplex Genome Engineering

The application of CRISPR/Cas systems to edit multiple sites in the genome simultaneously, as reported in $2013$.

Precision Repair Pathway

A term describing Homology Directed Repair (HDR) due to its accuracy compared to NHEJ.

Error-prone Repair Pathway

A term describing Non-homologous end joining (NHEJ) due to its tendency to cause mutations.

Ancestry Analysis Percentage

$17.00\%$ and $18.00\%$ are specific quantitative data points shown in the biotechnology application examples.

Transplantation of Healthy Cells

An application of iPSC technology where genetically matched healthy cells are returned to the patient.

Target mRNA in Tissue

The specific molecule localized during in-situ hybridization.

Secondary Antibody Conjugation

The process of attaching a fluorophore marker to a secondary antibody for signal detection.

Drosophila Primordial Germ Cells

Cells identified in the embryo using an antibody targeting the Vasa protein.

Mutagenesis Incidence

The use of chemicals or radiation to increase the rate of mutations in a classical genetic screen.

Snapdragon Mutant

An example organism used to demonstrate the effects of mutagenesis in classical genetics.

Double-stranded RNA (dsRNA)

The molecule expressed in E. coli that is processed into siRNA by C. elegans.

Post-transcriptional Degradation

The mechanism of RNAi where the target mRNA is recognized by base-pairing and destroyed.

Bacterial Immunity

The original natural function of the CRISPR/Cas system against viruses.

Clustered Regularly Interspersed Short Palindromic Repeats

The full descriptive name for the CRISPR locus.

Protospacer Requirement

The specific viral sequence that must be captured by bacteria to facilitate future CRISPR-mediated defense.

 Cas9 tolerate in most organisms

A characteristic of the Cas9 protein that allows it to function effectively across the kingdom of life.

Plasmid for Cas9 Expression

A circular DNA molecule used to introduce the Cas9 gene into a target cell.

Off-target Sequence Homology

The reason Cas9 might cut at unintended sites if they are similar to the guide RNA sequence.

Repair Template Homology

The requirement that a user-defined DNA molecule must match the sequences surrounding a Cas9-mediated cut.

Oncogene Creation Risk

A safety concern regarding off-target CRISPR cutting in human therapies.

Base Editing Safety

A method considered safer because it does not require double-stranded breaks in the DNA.

In Vitro Fertilization Controversy

The contentious use of CRISPR/Cas9 to edit human embryos in $2018$ to prevent HIV transmission.