Utility of Molecular Biology

Transcriptome:

Collection of all mRNAs expressed by a cell

In situ hybridization (individual gene):

examine location of mRNA and amount in a cell or tissue

RT-qPCR (individual gene):

measures relative levels of mRNA from a cell or tissue

RNA Seq (the entire transcriptome):

determines ALL the mRNAs present in a cell

In situ hybridization takes advantage of

complementary base pairing to determine where a gene is expressed in a cell or tissue

The DNA or RNA PROBE is marked (fluorescence or dye) and then

hybridization occurs in the tissue sample

Advantages of in situ hybridization

Examines localization of cells expressing a particular mRNAs

Disadvantages of in situ hybridization

• Must have a “fixed” sample

• Need to know the mRNA sequence

• Limited to only a few mRNAs at a time

When do we use in situ hybridization

When we want to see where a gene is expressed (ie is making mRNA) in an organism

RT-qPCR stands for

Reverse transcriptase, quantitative PCR

Advantages of RT-qPCR

• Fast, relatively cheap

• Can be used to determine relative mRNA concentration

Disadvantages of RT-qPCR

• Need to know the mRNA sequence

• Limited to only a few mRNAs at a time

When do we use RT-qPCR

When we want to know if a specific gene is expressed in cells

RNA Seq is used to

determine ALL genes are expressed and relative levels from a cells or tissue

Advantages of RNA seq

• Don’t need to know the sequence before the assay

• Learn about what isoforms are expressed in a cell

Disadvantages of RNA seq

• Expensive

• Generates a LOT of Data

When do we use RNA seq

When we want to know ALL the genes expressed in the cell

Methods for breaking a gene

• Knockdown the gene by destroying the mRNA

• Knockout the gene from the genome

• Generate mutants in the gene

RNAi:

use siRNA mechanism to target your mRNA for destruction. Knockdown the gene by destroying the mRNA

Homologous Recombination:

replacing coding sequence with a selectable marker. Knockout the gene from the genome

CRISPR:

create indels with a DNA break followed by NHEJ. Knockout the gene from the genome

Cloning:

express from a vector. Generate mutants in the gene

CRISPR:

create changes to the genome with homology directed repair (HDR). Generate mutants in the gene

RNAi technology advantages:

• Relatively cheap

• Easy to give to cells

• Does not require manipulation of the genome

RNAi technology disadvantages:

• Not a permanent (short-lived)

• Not a complete knock-out (may have residual expression)

When do we use RNAi Technology

• When we want to eliminate the mRNA/protein to learn about its function

• Only eliminates mRNA/protein not the gene in the organism’s genome

in RNAi technology instead of “foreign” RNA, we are going to introduce

dsRNA of our choosing

Bacteria (E. coli) contain a plasmid with a short stretch of DNA for

your gene of interest and they generate dsRNA from this plasmid

Double stranded DNA breaks are repaired in one of two ways

1. Nonhomologous End Joining (NHEJ)

2. Homologous recombination (HR)

Nonhomologous End Joining (NHEJ)

• The cell tries to quickly repair the break before the two fragments drift apart

• Error prone process (loss of nucleotides at repair site)

Homologous recombination (HR)

• Homologous DNA can serve as template for repair

• Can only occur if DNA break occurs shortly after DNA has been replicated

• OR if we give the cells DNA with homology

Use homologous recombination machinery to

“replace” the genomic DNA

Replace the gene’s coding sequnce with a

selectable marker

Supply the information to “repair” the region of DNA with a

selectable marker using homologous recombination

Only cells where this replacement has occurred will

survive drug treatment (like with bacteria and plasmids)

Eukaryotic Targeting:

Gene knock-out

NeoR

makes the cells expressing it resistant to G418.

tkHSV

cells expressing this convert the compound ganciclovir to a toxic compound - kills cells expressing it in the presence of ganciclovir - cells NOT expressing TK are resistant to ganciclovir.

Combination of NeoR and tkHSV

Permits selection of cells that have properly generated the knockout.

Steps for gene targeting in mice

1. Gene targeting in ES cells

• Isolate and culture embryonic stem (ES) cells from mouse blastocysts.

2. Construct targeting vector

• Create a DNA vector containing:

  • Homologous DNA matching the target gene

  • neoR gene → positive selection

  • HSV-tk gene → negative selection

3. Transfect ES cells

• Introduce the targeting vector into ES cells.

• Homologous recombination replaces part of the normal gene with the altered gene.

4. Positive-negative selection

• Add drugs to select correct cells:

  • neoR+ cells survive (they incorporated the vector)

  • HSV-tk+ cells die in ganciclovir (random insertion)

5. Expand targeted ES cells

• Grow the rare correctly targeted ES cells into colonies.

6. Inject into early embryo

• Inject altered ES cells into a mouse blastocyst.

7. Implant into pregnant mouse

• Place embryo into surrogate mother.

8. Generate chimeric mice

• Offspring contain both normal and altered cells.

9. Breed mice

• Breed chimeras to obtain mice carrying the knockout gene in all cells.

Key idea:
Correct knockout cells are usually neoR⁺ / tk⁻

ES cells are pluripotent so they can

contribute to all cell types in the mouse (including the sperm and egg)

ES cells are not totipotent since

they can not direct an entire organism - only that initial fertilized egg is totipotent

Typically use cells from mice of different coat color to determine

mice with integrated gene target

Mate the chimeric (mix of normal and targeted cells) mice until you have

mice only with the targeted gene

Mice expressing a mutant form of a DNA helicase exhibit symptoms of

premature aging

We can model disease in organisms to

study them

A mutant mice can mimic human mutation that causes trichothiodystrophy,

a disorder characterized by abnormalities that can reduce lifespan

This system functions in bacteria and acts like an “immune” system to target and destroy invading viruses

CRISPR-Cas system

• Bacterial defense mechanism against viruses (bacteriophages)

• Stores pieces of viral DNA as a memory of infection

• Uses guide RNA and Cas proteins to recognize and cut invading viral DNA

Scientists harnessed this CRISPR technology to:

• Generate gene knockouts to study gene function

• Or to precisely and permanently alter the genome of an organism

CRISPR works in

every cell type that it has been tested in, even cells that homologous recombination never work well in

We have the power to

alter rapidly, cheaply, and easily any genome (even humans…)

The CRISPR-Cas 9 system:

• Guide RNA is synthesized in the lab for our particular target and expressed in cells

• Complementary abse pairing, directs the Cas9 protein to a PRECISE site in the genome

• Cas9 is a nuclease and generates a double strand break in the DNA

For a gene knockout,

we hope that the NHEJ repair will insert or delete nucleotides (called indels)

For a gene knockout, we hope that NHEJ repair will insert or delete nucleotides (indels). What will this do to the resulting protein?

Indels often cause a frameshift mutation, which changes the reading frame of the gene. This usually leads to a completely different amino acid sequence downstream and frequently introduces a premature stop codon, producing a truncated, nonfunctional protein (effectively knocking out the gene).

For generation of a mutation (or repari of a mutation!) or insertion of DNA (hello GFP!) we want

homologous recombination or sometimes called homology directed repair (HDR) to replace the gene with the mutant. Must supply a donor template