mouse genetics recap

Question 1

how can you do engineered defined changes in mouse genomes?

Answer 1

• removing gene sequences - targetted mutations

- adding new gene sequences - transgenes

Question 2

what is gene replacement?

Answer 2

-only mutant form of gene present

Question 3

what is a gene KO?

Answer 3

no active form of a gene is present

KOs done to determine function

Question 4

how do you begin a KO project?

Answer 4

• genomic clone of your gene

- starting construct has neo inserted in target gene (destroy gene activity and TK off to one side

Question 5

where is the construct introduced to?

Answer 5

• mouse ES cells
• cells DNA machinary recombines construct into the mouse genome
• when homologous combinations occurs the TK gene is lost

Question 6

how can the KO be identified?

Answer 6

• double selection
• cells which have integrated the neo gene can grow in a neomycin containing medium (resistant to neomycin)
• cells which still have the tk gene will die in GANC media
• only cells left are KO cells as have neo but not tk - homologous recombination

Question 7

what happens to the selcted KO cell line?

Answer 7

it reintroduced into mouse embryos

• first generation moaic and gonads mosaic (mixture from mother and stem cell line)
• bred to generate non-mosaic carriers of the KO
• carriers are interbred to creat homozygous mutant animal

Question 8

what does crispr stand for?

Answer 8

clustered regulalry interspaced short palindromic repeats

Question 9

how does crispr work?

Answer 9

• double stranded DNA break made
• cellular repair processes kick in and we convince process to make the edit that we want(not a natural edit)
• CRISPR goes along the genome till it finds a match and inserts and rips strands apart- tirggeres cas9 to cut
• cells repair mechanism shoves peices back together - gets lost - creates a KO
• can edit live cells