RR16: Molecular biology of gene targeting Flashcards
How can we best address what the function of a gene is?
By removing the gene activity and analyzing the phenotype.
What do abnormal phenotypes after removing the activity of a gene tell us?
It means that there are specific processes that have been disrupted that were relying on that gene activity that was removed.
How can we remove the gene activity?
Disrupt homeostasis based on random mutations.
Disrupt the activity of specific gene product.
How do we call the disruption of homeostasis based on a random mutation?
It’s forward genetic analysis.
Meaning that we are not looking for a specific gene, we’re just looking for mutations in the phenotypes. When we have a mutant phenotype, we go back and see which gene was disrupted.
How do we call the disruption of the activity of a specific gene product to assess its function?
Reverse genetic analysis.
We’re interested in what the gene does, we remove the gene and see the phenotypes that arise from the removal of that specific gene.
When we observe the phenotypes of all the genes that had unknown functions in the genome of C. elegans, what was that removal of gene activity called?
It was reverse genetic analysis.
The RNAi were for a specific gene, and each bacteria possessed a dsRNA that was different for each C. elegans. We were looking for what the phenotypes were when we took out specific genes.
What is gene targeting?
It’s used in bigger organisms than C. elegans, more used in mice for example.
It’s used to change a specific sequence or a gene in a genome.
It’s like reverse genetic analysis but for bigger organisms.
Can we use homologous recombination in mice?
Yes. Homologous recombination events in mice happen in the embryonic stem cells.
How does homologous recombination work in mice?
- Have a longer homologous sequence, so a lot of flank sequences. Need to know more about the segment you want to replace.
- Have a drug-resistant gene
- Make a disruption construct with the flank sequences and the drug-resistant gene
- Have thymine kinase, a gene outside the gene we want to do the homologous recombination.
- Thymine kinase is a herpes simplex virus kinase that will metabolize a specific drug, so cells can’t live. we do this to make sure we get a direct homologous recombination event, not a random insertion.
- Transfect that into ES cells
- Use the drug to get rid of all the cells that didn’t integrate the construct.
What are embryonic stem cells?
ES cells.
They are pluripotent, so they can give rise to several different cell types.
When we do homologous recombination in mice, we can get 2 alternatives on how the recombination takes place. What are the 2 options?
Direct homologous recombination insertion. So we just exchange the gene of interest and put in our mutant gene with the drug-resistant in. (the thymine kinase doesn’t go in the new gene)
OR
Random incorporation of the entire chunk of DNA. We just get the entire gene region inserted with another gene region. That’s why we have the Thymine kinase, to make the cell non-viable when we don’t get direct homologous recombination.
How can the thymine kinase be toxic to the cells?
By putting all the cells in Ganciclovir.
Ganciclovir is toxic in the presence of the thymine kinase gene, so it will kill all the cells that didn’t get the direct homologous recombination. It kills the cells that got random insertions.
When doing homologous recombination in mice, we use 2 rounds of selections to make sure we only analyze the cells with the mutant gene. What are those 2 rounds of selection?
- Using Ganciclovir.
It will kill all the cells that did a random insertion and added Thymine kinase. Ganciclovir and thymine kinase together become toxic for the cell and kills it. - Using G-418.
It will kill all the cells that didn’t take up our mutant gene with the drug-resistant in it. It eliminates the cells that stayed normal or that didn’t take up the gene correctly.
That way, we’re only left with ES cells that have undergone homologous recombination.
How do we inject that mutant gene we got from the homologous recombination into a real mouse?
The mutant gene is in embryonic stem cells. That means that those ES cells can give rise to all the tissues in the mouse.
1. Inject the ES cells (of a brown mouse) in the blastocyst of a black mouse
brown is dominant, black is recessive
2. The cells mix in early embryo and they divide.
3. The blastocyst gets put into a mom that will give birth to 2 types of mice: black and chimeric mice.
4. The black mice are like the mom, they’re not modified.
5. The chimeric mice are modified, they have brown and black coat, so it means that the initial cells chose black and brown chromosome in different cells giving rise to chimeric mice.
6. Hope that the cells that decided to chose the chromosome with the brown dur are the germ cells to be able to cross them again.
7. If the germ cells are affected by the mutant gene, you keep going until you got homozygous pure manipulated brown animal.
8. You have to act fast to figure out what gene is affected because they will die. And then, it doesn’t give us much information.
9. Or you can do all that and the animals turn out fine, so you don’t know anything then.
Not the best way
What’s a better way to figure out gene function in mice, other than doing homologous recombination in ES cells?
Transgenic mice.
They are simpler to make than doing homologous recombination on ES cells and they can provide important information.
What are transgenes?
A gene that is artificially introduced into the genome of another organism.
How can we use transgenes to modify the phenotypes of mice?
It’s based on the idea that we often get random integration events when modifying a gene.
- Make a transcriptional fusion and introduce it in an embryo
- That embryo will become a mouse that expresses that transcriptional fusion gene
- Observe what that transcriptional fusion gene modified on the phenotype
OR - Take a gene product and make it into a dominant negative gene product by removing DNA-binding domain
- Introduce the transgene without the DNA-binding domain
- See how the phenotype expresses the gene. Usually, we lose the expression of that gene because it lacks the DNA-binding domain, so we see how the phenotype is without the expression of that gene.
What are transgenes used for?
They can modify the genome.
They integrate randomly into the genome.
Help us understand the expression patterns of genes.
Can express transgenes under endogenous promoter (promoter upstream if the gene) or heterologous control (control that is not normally in the gene, like inducible promoters (heat, heavy metals…)).
