Genetic manipulation 2 Flashcards

1
Q

MsX1 deficient mice

A

cleft palate and craniofacial/tooth abnormalities

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2
Q

2 licenses required for genetically modifying animals

A

project license

personal license

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3
Q

Act for project license when working with GM animals

A

Animals (scientific procedure act) 1986

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4
Q

4 reasons for genetic manipulation

A

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

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5
Q

3 examples of spontaneous mouse mutations

A

small eye - Pax 6
looptail - vangl2
clubfoot - limk1

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6
Q

Random mutagenesis - how is it done?

A

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

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7
Q

examples of agents used in random mutagenesis

A

radiation
ENU
EMS

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8
Q

ENU - what does it do?

A

creates point mutations by ethylating DNA base pairs in replicating sperm

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9
Q

EMS - what does it do?

A

turns G/C into A/T

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10
Q

screening for dominant mutations

A

male mated with wild type female

heterozygous = phenotype

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11
Q

screening for recessive mutations

A

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

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12
Q

uses of random mutagenesis

A

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

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13
Q

disadvantages of random mutagenesis

A

use large numbers of animals

wasteful - increasingly hard to justify

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14
Q

basis of gene knockouts

A

delete gene in ES cells and inject into blastocyst

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15
Q

homologous recombination - when does it usually occur?

A

meiosis

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16
Q

briefly explain what homologous recombination is

A

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

17
Q

what can homologous recombination be used for?

A

to introduce new DNA into cells

18
Q

Important note about flanking DNA sequences

A

transgene has identical flanking DNA to sequence that normally surrounds DNA

19
Q

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

A

fluorescent green protein

neomyicin resistance

20
Q

After electroporating cells explain how homologous recombination will work

A

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

21
Q

After homologous recombination what ES cells should be selected?

A

ganciclovir - cells with TK are killed

neomycin - cells without neoR are killed

22
Q

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

A

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

23
Q

What % of integration events does hr account for?

24
Q

1 in ? cells undergo homologous recombination

A

10 million

25
1 in ? cells come through +ve/-Ve selection will turn out to be correctly targetted
200
26
Give an example where redundancy may lead to mild or no phenotype
myoD
27
Example of early embryonic lethality preventing analysis of later events
oct 4 or fgf 4
28
what else may affect the interpretation of knock outs?
genetic background and strain of mouse
29
knock in
the use of targeting vector and homologous recombination to introduce a new functional gene at a known location in a genome
30
use of knock ins
reporter mice expressing Green fluorescent protein | expressing one gene under control of a promoter of a different gene
31
therapeutics from animals - gene knock in
b - interferon = important in therapeutic | green fluorescent milk...