L11 Transformation in Mouse

Question 1

Transformation in mouse

Answer 1

Initial experiments - microinject DNA directly into the embryo, very inefficient

Major advance - development of ES cell cultures
Embryonic stem (ES) cells:
- can be maintained in culture
- form colonies (unlike most animal cells)
- remain in undifferentiated states
- but pluripotent => retain the ability to differentiate into different cell types

Question 2

Transformation of ES cells by electroporation

Answer 2

See onenote diagram

• to get DNA into any cell
• strong electric pulse opens the cell membrane pores
• uses a selectable marker e.g. neomycin resistance
• fate of DNA = must be integrated to be maintained (rate or non-homologous integration much greater than homologous integration)

Question 3

Micro-injection of ES cells into blastocyst

Answer 3

See onenote

• ES clones from brown mouse inserted into blastocyst of white mouse
• injected into blastocyst of a white mouse
• implanted into pseudo pregnant female

Question 4

Selecting transgenic mice

Answer 4

See onenote slide

• implant embryo into pseudo pregnant female
• chimera = successful integration, brown patches derived from ES cells, evidence that it has been transformed with DNA

Question 5

Gene transfer in the mouse - summary

Answer 5

• labour intensive and low frequency
• DNA must be integrated to be maintained
• frequency of non-homologous integration much greater than homologous integration

Question 6

Targeted disruption of HPRT gene in mice

Answer 6

See onenote

• knockout mice
• selection for homologous recombination possible but two caveats:
  1. hprt- have a selectable phenotype in ES cells
  2. hprt gene is x-linked, used male ES cell lines

Question 7

Generalised strategy for gene targeting in ES cells

Answer 7

See onenote

Positive-negative selection
- increases identification of homologous integration

Positive = select for transformants e.g. NeoR (G418)

Negative = select against non-homologous integration events e.g. HSV-TK (herpes simplex virus thymidine kinase) (GANCs, GANC sensitive)

Question 8

p53 gene

Answer 8

• role in cell cycle
• alteration to p53 gene associated with many cancers
• due to loss-of-function mutations? over-expression/ altered function? null mutation?

Question 9

Targeted knockout of p53 in mouse

Answer 9

See onenote slides

If P53 is an essential gene, there should be a distortion in the Mendelian ratio BUT 1:2:1 is normal => p53 is not an essential gene

p53null/p53null viable and developed normally - dispensable to development BUT…

• dramatically increased frequency of cancer so loss of p53 => malignancy
• suggests p53 has a protective role, recognised as a tumour suppressor

Question 10

Role of p53

Answer 10

• p53 protein similar to known transcription factors
• does p53 activate expression of genes that prevent cancer or does p53 repress expression of genes that cause cancer?
• use yeast determine whether p53 can function as an activator of gene expression

Question 11

GAL4 TF

Answer 11

• regulates galactose breakdown in yeast

- GAL4 has a DNA binding domain and an activation domain, the two domains can be separated

Question 12

Is p53 an activator?

Answer 12

See onenote slides

• replace GAL4 activating domain with p53, the DNA binding domain of GAL4 stays the same
• will p53 activate expression of reporter gene, lacZ?
• transformed into yeast
• expression occurs, p53 has activator function in yeast
• p53 known as “guardian of the genome”, a TF factor that activates DNA repair

Question 13

Mouse transformation summary

Answer 13

See onenote summary page

Question 14

Various strategies developed to expand the possibilities available in the mouse

Answer 14

1. Knock in - gene of interest expressed in place of target
2. Cre/loxP - to introduce specific mutations, inducible
3. double replacement - creating clean mutations

Question 15

Generating knock-in

Answer 15

See onenote slide

e.g. put reporter into locus of gene to understand its expression

Question 16

Using cre/loxP to alter genomic sequences

Answer 16

See onenote slide

• cre recombinase from bacteriophage P1 - site specific recombination between 34bp loxP sites
• depending on the orientation of the loxP sites, you get a different outcome e.g gene inversion, gene deletion

Question 17

Using cre/lox to remove genomic sequences

Answer 17

See onenote slides

Question 18

High-throughput discovery of novel developmental phenotypes

Answer 18

See onenote slides

Question 19

Cre/lox introduce specific mutations

Answer 19

See onenote slides

- create subtle mutation, not a null

Question 20

Introducing “clean” mutations in the mouse genome

Answer 20

• double replacement (not yeast two-step gene replacement)

1. introduce a selectable marker into the genome at the required locus
2. replace the selectable marker with the specific mutation you wish to introduce into the genome

Question 21

Double replacement

Answer 21

See onenote slides

- mutated genome, no trace of vectors