Lecture 3 Flashcards
How can we grow embryonic stem cells in lab while limiting the cell differation?
Minimize the time in cell culture, grow in special surfaces and feed with fetal bovine serum and use leukima inhibitor factor.
How can homologus recombination be used to cause a gene knockout?
Breaking the DNA at both strands the cells repair mechanism will try to repair the DNA but often unsuccessfully and the gene sequence will be altered and no longer functional or maybe not there at all.
What is TAG exchanged, how is it used and what are the disadvantages?
It uses two homologus recombination steps, first tagging a gene sequence with a selectible marker and in the second step the tagged sequence being replaced by a homologus sequence with certain mutations. But it requires two steps and can have a high failure rate due to the colonies being damaged.
What is a selectible marker?
It’s a gene sequence that can be seen or detected with the function to show if the succes of transduction and is often antibiotic resistant genes.
What types of markers are there and how do they work?
Positive markers has a positive effect for the organism for example antibiotica resistance. Negative markers eliminate or inhibit cell growth. Positive and negative markers can have a both positive and negative effect on the cell depending on the enviormeant.
How do you perform gene targeting in a lab?
Use the correct vector and homologus recombination.
What is Cre Lox recombination and how is it used?
It’s a site specific recombination technology and can target modification to a specific cell type or to be triggered by eternal stimulus. The cre recombinas enzyme recgonise two specific lox in the DNA and causes recombination between them. It can cause gene deletion, gene rotation or gene slecivity.
What check points is there for coning?
Check that the genetic modification is in the correct location, that no unintended recombination has occured, verify vector and perform PCR for first screening.
How do you screen for mutants?
Seperate organisms based on a specific phenotype, insert a negative marker and do genotyping for the selected region.
How does zink finger nucleases work?
It can target and alter highly specific sites in the genome using the cells DNA repair function.
What are Transcription activator-like effector nucleases (TALENs) and how do they work?
Using restriction enzymes it can cut and bind gene srquences
What is CRISPR/CAS9 and how does it work?
Using the CAS9 protein and a guide RNA the genome can be edited.
