Transgenic organisms and knockouts 1 & 2 Flashcards
What’s the difference between forward- and reverse genetics?
Fw (You start at the front): After having achieved a phenotype of interest, you try to find the genotype.
Rev (You start at the back): Mutate the genotype, and assay for the phenotype of interest.
What are three ways of inducing mutations in a random way, good for forward genetics?
Ionization
Chemicals - Example: EMS (Ethyl methanesulfonate), alkylates DNA –> missense mutations.
Transposons
What’s a great advantage of forward genetics using transposons to induce mutations?
You know the sequence which is transposed, this acts like a molecular tag. In order to find where the transposon has entered the genome, design primers for it and run an inverse PCR.
Briefly describe the two transposons used for forward genetics.
Sleeping beauty: Defect and relatively inactive. The transposon can make local jumps.
piggyBac: More active than sleeping beauty, makes global jumps. Preference for intragenic regions.
How can the Ti-plasmid from agrobacteria be used to grow transgenic plants?
The Ti plasmid needs contain 5 domains apart from the T-DNA of your choice:
Bacterial selection marker
Cauliflower Mosaic Virus 35S promoter / terminator
Bar gene (basta resistance, a certain pesticide)
Bacterial origin of replication.
Procedure
Transform bacteria with Ti-plasmid, then grow the bacteria on a selection medium fitted for the selection gene.
Take discs of tobacco leafs and incubate them on agrobacterium plate with a selection pesticide medium (Basta) (only plant cells with T-DNA will proliferate).
Switch to shoot-inducing medium, allow tobacco shoots to grow.
Transfer shoot to root-inducing medium
Grow tobacco plant, which is now transgenic.
In order to find and prove that the plant which has grown is a transformant, we can verify the Ti-DNA sequence in different samples taken from the plant. This can be done by PCR or DNA-sequencing.
How can genes in embryonal cells be knocked out using homologous recombination?
Embryonic stem cells (general KO!). See figure 1 and figure 2 for extra details.
cells are extracted from embyoblast and allowed to replicate.
Target DNA construct is created containing neomycin resistance.
Transfection via electroporation.
Cell - vector do homologous recombination, exchanges GOI with neomycin resistance.
Selection of good recombinants. There will be 90% false positives where homologous recombiantion have partially occured. Southern blot / PCR / Seq for true positives.
Counter-selection of a gene product which shouldn’t remain if homologous recombination has occurred, and not NHEJ. See figure 1.
Plant selected stem cell in surrogate mother to generate a chimera, these are also referred to as founder mice.
This KO-method relies on a visible, selctible phenotype (which is due to a dominant mutation). Mate with wild type, select heterogenous profeny.
Inbreed two of the heterogenous progeny.
Achieve fully transgenic mouse.
Describe how the cre-loxP system can manipulate the genome.
Cre-LoxP (Specific KO, inversion or substitution). Benefit: You can KO tissue-specifically. A benefit of the cre-loxP system is that you can generate conditional knockouts in different tissues or other circumstances. The loxP sites are 34bp and can be placed in different polarities to achieve different outcomes (inversions, duplications, deletions, translocations).
Cre recombinase is engineered into a mouse genome downstream of a known promoter via ESCs as discussed above.
Second mouse contains the target gene flanked by 2 loxP sites. Depending on directionality, different outcomes occur. Also engineered into the mouse genome via cre-loxP.
Cross the mice. Select mice w/ visible phenotype / sequencing.
What’s the analogue to cre-loxP in yeast?
FLP-FRT
FLP = Flippase
FRT = Flippasse recombinase target sequence
How can the P-element be used to generate transsgenic drosophila?
You can generate transgenic drosophila using the P-element, which is a naturally occurring transposase. Males host the transposase, females can produce a P-transposase inhibitor from the same genetic element, allowing for genomic stability and possible progeny. When utilizing the male P-element in the lab, you transfect one plasmid hosting your GOI flanked by P-transposition sites (+ marker gene). The second plasmid hosts the transposase. Both of the plasmids are transfected into the drosophila embryos.
How do Tetracycline-inducible promoters work?
- Incorporate the tetO element in between your GOI and its promoter.
- Grow on tetracycline-infused plates. Tetracycline binds the TetO element and acts like a transcriptional repressor.
- Add doxycycline, it disinhibits the repression by binding tetracycline.
How do Tamoxifen-inducible promoters work?
- Insert your gene of interest downstream of the estrogen receptor element, ensure that the gene product will be a fusion protein.
- Grow on HSP90 infused plates. HSP90 will inhibit the ER-GOI complex.
- Administer tamoxifen. It dissociates the ER-GOI complex from HSP90, making it active.
How does Zinc finger nucleases work?
A zinc finger protein contains one or multiple fingers which host DNA specificity. The zinc finger nucleases cleave based on this specificity.
(add more from methods course if there’s time)
A: How does CRISPR-cas9 work?
B: How can you modify the system for transcriptional repression?
C: How can you make cas9 more specific?
- gRNA binds cas9
- cas9 is guided by gRNA to genetic code which is to be cleaved.
(3A) Add repair template in order for homologous recombination to take place and create a KO w/ marker gene.
(3B) Catalytically kill the cas9 enzyme in order to make it transcriptionally repress the target, if you want to.
(3C) Make so that cas9 cleaves ssDNA, add two cas9s to increase specificity.
How does cas9 prime editing work?
Prime editing is when cas9 utilizes a pegRNA instead of a gRNA.
- The cas9 enzyme generates ssDNA break at a location complementary to the pegDNA (the genetic code you’d like to mutate).
- The pegDNA acts as template for RT so that its mutation gets incorporated into the host genome. ( The pegRNA has a 3’ overhang outside of its complementary sequence which acts like a substrate for RT.)
- Flaps will be generated by RT when it elongates the complementary strand along the pegRNA scaffolding. These are removed by cellular exonucleases.
- cas9 induces a break in the antisense strand at the complementary location. It will be repaired by ssDNA repair machinery w/ the sense-strand as a repair template.
How do TALENs work?
Two TAL proteins fused w/ fok1 are designed to bind in close proximity to each other so that fok1 can dimerize and cleave.
Requires a lot of engineering.
