LECTURE 17!

Question 1

examples of model organisms

Answer 1

mouse, clawed frog, sebrafish, fruit fly + nematode worm

Question 2

why use mice?

Answer 2

share 95% genes, short life cycles

Question 3

what are ES cells?

Answer 3

embryonic stem cells

Question 4

properties of in vitro models

Answer 4

not representative of a protein’s role in a biological system like a body

Question 5

what are the mouse models to study human development and disease?

Answer 5

transgenic, knock-out + knock-in mice

Question 6

what is the standard transgenic approach?

Answer 6

DNA injected into pro-nucleus of mouse oocyte
transferred to a pseudo-pregnant recipient mouse
ALL offspring screened for expresion of transgene by DNA analysis

Question 7

what is gene-targeted transgenic approach?

Answer 7

• isogenic transgene with drug selection gene introfuced to embryonic stem cells
• drug selection used
• surviving cells screened for correct intergration of transgene
• correct cells injected to mouse blastocytes
• transferred to a pseudo-pregnant recipient mouse,
• chiameric offspring identified and mated to test for germline transmission of transgene

Question 8

what does a transgenic approach require?

Answer 8

gene of interest, relevant promoter, 3’ protein tag for detection + poly A tail

Question 9

what are gene traps?

Answer 9

used to introduce insertion mutations across the genome in mouse embryonic stem cells

Question 10

insertion of a gene trap vector…?

Answer 10

distrupts + reports gene expression and prvides a convenient tag for the identifcation of the insertion site

Question 11

typical ‘genetrap’ from interanational mouse phenotyping consortium

Answer 11

to provide targeted inactivation through recombination using FRTand loxP

Question 12

what do FRT and loxP sites mediate?

Answer 12

site-specific recombination through DNA recognition sites

Question 13

what is cre recombinase?

Answer 13

tyrosine recombinase enzyme, catalyses site-specific recombination between two LoxP sites

Question 14

what is flippase?

Answer 14

tyrosine recombinase enzyme, catalyses site-specific recombination between two FRT sites

Question 15

loxP sites allow…?

Answer 15

straight forward knocks-outs to be generated

Question 16

FRT and loxP sites allow…?

Answer 16

conditional knock outs to be generated

Question 17

strengths vs weakness of knockout mouse models

Answer 17

‘genetraps’ available for virtually all genes, can make conditional KO use cre recombinase, can stress or cross onto a different background, may not accurately model a known human disease

Question 18

knock in technology main use

Answer 18

to introduce a human mutation into the mouse gene

Question 19

what is function of CRISPR cas9?

Answer 19

-utlisies short RNA molecules with DNA cutting enzyme to protect against viral infection