Genetic Manipulation 4

Question 1

What are the 2 ways around the problem of a mouse dying due to gene knockout in all cells?

Answer 1

create chimeric mouse

cre/loxp to make tissue specific knockout

Question 2

what is cre?

Answer 2

a site specific recombinase enzyme from the p1 phage

Question 3

what does cre recognise?

Answer 3

a 34bp DNA sequence

Question 4

what is used instead of cre and lox p in mice?

Answer 4

flp recombinase from S. Cerevisidae which recognise FRT

Question 5

what is needed for cre/loxp in terms of strains of mice?

Answer 5

need 2 strains

Question 6

what happens in 1st strain of mice in cre/loxp?

Answer 6

loxp inserted around gene of interest = floxed

Question 7

what happens in 2nd strain of mice in cre/loxp?

Answer 7

express cre recombinase from a tissue specific promoter

Question 8

what happens when the 2 strains of mice are bred together in cre/loxp?

Answer 8

gene knocked out only in tissue of interest in which cre is expressed

Question 9

briefly summarise how the floxed mice is made

Answer 9

homologous recombination in ES cells - electroporation
inject into blastocyst = chimeras and breed
neoR removed to give floxed allele

Question 10

drive cre from promoter of a gene that (3)

Answer 10

is only expressed in tissue you want (rare)
is expressed in areas that overlap with your tissue
designer promoter - drive expression of other genes in your tissue of interest

Question 11

IRES

Answer 11

internal ribosome entry site

Question 12

what has historically been the rate limiting step of cre/loxp?

Answer 12

strong cre expression only in places where you want it

Question 13

how to knock in a cre gene?

Answer 13

HR in ES cells

Question 14

2 drugs in which the promoter the cre gene is on can be responsive to

Answer 14

tetracycline

tamoxifen

Question 15

tet-off systems - briefly describe

Answer 15

tet A protein bend to tet O to activate cre transcription when no tetracycline, tetracycline blocks this

Question 16

What is tamoxifen induction of cre expression?

Answer 16

fusion protein combining activity of cre and a mutant form of ligand binding domain of oestrogen receptor

Question 17

does cre-ER bind oestrogen?

Answer 17

no

Question 18

what does cre-ER bind?

Answer 18

4-OH-tamoxifen

Question 19

what happens to cre-ER if no tamoxifen?

Answer 19

Hsp90 grabs cre-ER and keeps it in cytoplasm so no recombination in nucleus

Question 20

what happens to cre-Er if tamoxifen present?

Answer 20

cre-TRM released from Hsp90 and recombination