Genetic manipulation 2

Question 1

MsX1 deficient mice

Answer 1

cleft palate and craniofacial/tooth abnormalities

Question 2

2 licenses required for genetically modifying animals

Answer 2

project license

personal license

Question 3

Act for project license when working with GM animals

Answer 3

Animals (scientific procedure act) 1986

Question 4

4 reasons for genetic manipulation

Answer 4

to understand genetic basis of human health and disease
To identify and analyse roles of genes - over expression and knockout
understand control of gene regulation
genetically tag animals/designer animals for disease models

Question 5

3 examples of spontaneous mouse mutations

Answer 5

small eye - Pax 6
looptail - vangl2
clubfoot - limk1

Question 6

Random mutagenesis - how is it done?

Answer 6

male subjected to mutagen and leads to randomly distributed point mutations at low frequency through genome

Question 7

examples of agents used in random mutagenesis

Answer 7

radiation
ENU
EMS

Question 8

ENU - what does it do?

Answer 8

creates point mutations by ethylating DNA base pairs in replicating sperm

Question 9

EMS - what does it do?

Answer 9

turns G/C into A/T

Question 10

screening for dominant mutations

Answer 10

male mated with wild type female

heterozygous = phenotype

Question 11

screening for recessive mutations

Answer 11

may not have a phenotype if heterozygous - breed litter of progeny then mate brothers and sisters

Question 12

uses of random mutagenesis

Answer 12

can generate mutations in tissues without prior assumption of important genes
generate new alleles of genes never made deliberately or thought of

Question 13

disadvantages of random mutagenesis

Answer 13

use large numbers of animals

wasteful - increasingly hard to justify

Question 14

basis of gene knockouts

Answer 14

delete gene in ES cells and inject into blastocyst

Question 15

homologous recombination - when does it usually occur?

Answer 15

meiosis

Question 16

briefly explain what homologous recombination is

Answer 16

identical sequences on maternal and paternal chromosomes line up and cross over

Question 17

what can homologous recombination be used for?

Answer 17

to introduce new DNA into cells

Question 18

Important note about flanking DNA sequences

Answer 18

transgene has identical flanking DNA to sequence that normally surrounds DNA

Question 19

What can you replace the gene with in homologous recombination to make it more recognisable?

Answer 19

fluorescent green protein

neomyicin resistance

Question 20

After electroporating cells explain how homologous recombination will work

Answer 20

Over the next couple of days, most ES cells will expel or degrade any DNA they took up
A small proportion will integrate targeting vector into their genome randomly
A small proportion of those cells that integrate targeting vector will do so by Homologous Recombination

Question 21

After homologous recombination what ES cells should be selected?

Answer 21

ganciclovir - cells with TK are killed

neomycin - cells without neoR are killed

Question 22

after selecting ES cells post homologous recombination what is done next?

Answer 22

PCR and southern blotting on genomic DNA from survivors to check integrity of integrated vector

Question 23

What % of integration events does hr account for?

Answer 23

<2%

Question 24

1 in ? cells undergo homologous recombination

Answer 24

10 million

Question 25

1 in ? cells come through +ve/-Ve selection will turn out to be correctly targetted

Answer 25

200

Question 26

Give an example where redundancy may lead to mild or no phenotype

Answer 26

myoD

Question 27

Example of early embryonic lethality preventing analysis of later events

Answer 27

oct 4 or fgf 4

Question 28

what else may affect the interpretation of knock outs?

Answer 28

genetic background and strain of mouse

Question 29

knock in

Answer 29

the use of targeting vector and homologous recombination to introduce a new functional gene at a known location in a genome

Question 30

use of knock ins

Answer 30

reporter mice expressing Green fluorescent protein

expressing one gene under control of a promoter of a different gene

Question 31

therapeutics from animals - gene knock in

Answer 31

b - interferon = important in therapeutic

green fluorescent milk…