Lecture 28 (RR16): Molecular Biology of Targeting. Flashcards
What functions do genes have?
- The ideal way to assess the function of a gene is to remove its function and examine what that gene actually contributed to the overall phenotype.
- Function is best addressed through removal of gene activity and analysis of the resulting phenotype
- Abnormal phenotypes indicate specific processes have been disrupted that rely of the activity of the affected gene
Removal of gene activity
forward genetic analysis vs reverse genetic analysis
- Disrupt homeostasis based on random mutation -> forward genetic analysis
→ Forward genetic analysis: All starts with an interest in a phenotype. Introduce mutations randomly (using mutagenic chemicals or radiation), then you can recover mutants. You end up getting changes in a gene that give rise to a change in the overall organism, then you can work back and try to figure out what the gene was that was affected, which allows you to make a conclusion of what that gene actually does for that cell. - Disrupt the activity of specific gene product to assess its function -> reverse genetic analysis
→ Reverse genetic analysis: Starts with an interest in a specific sequence. How that gene product affects the overall development of the physiology of an organism. Disrupt the gene function to assess how that particular gene might contribute to the overall physiology of that organism.
Functional Genomics
- C. elegans researchers “knocked-down” every predicted transcription unit on chromosome 1 by using feeding RNAi…
- …of the analysed genes, 339 were assigned some function as determined by the visible RNAi phenotype
- Later they did it for each predicted gene in the C. elegans genome (~19,000 genes).
- RNAi: investigators were able to understand something about all the gene functions within a organism (initially this was C elegans). They did this by generating individual plasmids constructs that would make an RNA following transcription of one strand of a gene and then another RNA following transcription of the complementary strand of the same gene. This is an inducible system to make 2 RNAs that come together within a single bacteria.
- Each one of the predicted genes in C Elegans were used to make individual plasmids that would make dsRNA that corresponded to each one of those individual predicted genes. You could use these to feed C elegans by allowing them to eat it on bacterial plates. By virtue of an amplification system that exist in C elegans, takes a little bit of dsRNA and generate lots of RNA.
- You can seen phenotypes that correlate to loss of function that correspond to each one of the dsRNAs that are generated by each one of those bacterial columns that corresponds to each one of the predicted genes.
- Allows you to understand the contribution of individual gene products in the phenotype of this organism.
- The same process Was used in flies, mice, cultured cells - carry out these reverse genetic analysis at genome wide level.
Modifying the yeast genome
- as long as you have 20 nucleotides (on either end) of homologous sequence attached to a dominant selectable marker, you can use this disruption construct to recombine and replace a locus on a specific chromosome, all due to the homologous recombination that is directed by those homologous flanking sequences.
- Select for those events by growing the yeast in the drug, and only those yeast that actually took on this construct and incorporated the proper locus will actually be able to grow.
- Then assess what those individual genes actually did in various context in the single cell.
HR can also occur in…
- Owen Smithies and Mario Capecchi originally showed that homologous recombination could also occur in pluripotent embryonic mouse cells (ES cells).
- The combined strength of this kind of genetic manipulation with the, at that time, newly characterised ES cell lines isolated from mouse embryos made this strategy extremely powerful
- Sir Martin Evans (ES cells), Owen Smithies, and Mario Capecchi (HR) were awarded the 2007 Nobel Prize for Physiology or Medicine
- They combined the idea of homologous recombination and these embryonic stem (ES) cells to carry out replacements of various locis in the chromosomes that are present in these embryonic stem cells (similar to pluripotent cells that are present in the Early in the embryonic mass of a mouse).
Understand this this is a homology driven insertion!!
The 2 different situations you are going to select for:
1) non specific insertion of your replacement construct somewhere in the chromosome (random insertion) —> confer some sort of survival in the presence of the drug
2) Directed homologous driven recombination event is going to take place and in doing so, you will exclude that little yellow square outside the homology (Gene targeted insertion)
How do you select against the non specific (random insertion)?
- If you treat with G-418, it will select for all recombination events (positive selection)
- Ganciclovir is toxic in the presence of the herpes virus tk gene (yellow block), so it will negatively select against all non-homologous recombination events. Block cells at DNA replication - they are killed. So, any chromosome with the thymine kinase gene will be killed. Get rid of all recombinants with random insertion.
- Only ES cells that have undergone Homologous Recombination (HR) can survive the two selection steps. They will be selected for because they don’t have the thymine kinase gene and will survive in the presence of ganciclovir.
- Homology driven recombination event replaced one section of a chromosome.
From cells to chimeric mice
- ES cells are then used to populate and blastocyst of an acceptor mouse (intoduce ES cells into a growing blastocysts). The ES cells have to have some sort or marker so that you can tell whether those ES cells are present (the ES cells in code for a brown coat colour that is dominant in the mouse). This mouse strain has to be another coat colour that is recessive (black).
- The blastocyst is transferred to a surrogate mouse mother
- The progeny will be a mixture of both genotypes if the cells were viable and the process worked properly.
- The population of totipotent cells is therefore heterogeneous in the blastocyst
- Homogeneous/homozygous host embryos will give rise to black mice
- Embryos that are heterogeneous (have cells from the host (black fur) and the targeted ES cells (brown fur) will be CHIMERIC ie…coat colour will be spotted/patched)
- This means that the implanted cells contribute to various tissues. The hope is that one of the tissues they contribute to is the germ line!
*Sometimes you wont get any animals they will just die (you hit an essential gene)
2 possible outcomes
This whole process is gene disruption by homologous recombination. It is lengthy, costly and sometimes does not give you the outcome you want.
Transgenic mouse
What is the role of CRISPR
CRISPR is a process that bacteria have aquired to fight off infections.
CRISPR steps
CRISPR (Clustered Regularly Interspaced Palindromic Repeats)/Cas system is a natural mechanism by which bacteria protect themselves against foreign DNA (e.g. phage DNA).
1) Bacteria cleaves the invading phage DNA and adds the segments into the CRISPR array.
2) Transcription of the array contains mRNA with CRISPR repeats and invading DNA.
3) Repeat sequence binds to tracrRNA which provides a scaffold for Cas protein.
4) When the bacteria gets infected again with that phage, the complex base pairs with the phage DNA.
5) Cas cleaves the phage DNA.
What did scientist do with CRISPR?
Researchers have adapted the system to be able to work in eukaryotic systems to cleave the genome at specific sites.
* Cas9 - engineered version of the Cas endonuclease.
* Guide RNA - engineered to bind to Cas9 and to a specific site on genomic DNA (site of choice).
- This site can be any gene the researcher chooses to inactivate or replace!
* When Cas9 and the guide RNA are both expressed in cells, Cas9 will cleave the DNA at the targeted site.
* The DNA will often repair itself through Non-Homologous End Joining, resulting in the loss of a few base pairs.
- Can cause a deletion or frameshift which inactivates the gene.
* Genes can also be introduced if the cell repairs itself through Homology Directed Repair (HDR) mechanism.
- Add segments that match the sequences flanking the cleavage site.
- When the DNA is cleaved, homologous recombination will insert the donor DNA sequence into the cleavage site during DNA repair.
