Mutagenesis and Gene Knockout in Animals

Question 1

What regulation exists for people wanting to experiment on animals?

Answer 1

All projects involving animals that fall under the Animals (Scientific Procedures Act) 1986 must be specifically approved by the Home Office ⟶ Project Licence

Everyone working on animals must have Personal Licence

Question 2

What is the purpose of gene mutation in mice?

Answer 2

Genes can be mutated in order to find out what they do

Question 3

Give examples of spontaneous mice muations

Answer 3

Small eye mouse = pax6 mutation

Looptail mouse = vangl2 mutation

The ‘pma’ mouse (clubfoot) = limk1 mutation

Question 4

What are some of the available mutagens for mice?

Answer 4

Radiation

ethylnitrosourea (ENS)

ethyl methanesulphonate

Question 5

How does ethylnitrosourea cause mutations?

Answer 5

ENU creates point mutations mostly by ethylating DNA base pairs during DNA replication in replicating sperm cells

Question 6

How does EMS result in mutations?

Answer 6

EMS mostly turns G/C base pairs into A/T during DNA replication

Question 7

What is the distribution of mutations when using ENU or EMS?

Answer 7

Causes randomly distributed mutations throughout the genome at a low frequency

Question 8

How do we create an offspring mouse to hold a dominant mutation?

Answer 8

Use a mouse that produces mutant spermatozoa. Mate with wild type female.

Screen babies for the mutation - those with who are heterozygous with the dominant mutation will show a phenotype.

Question 9

How do we achieve mice containing recessive mutations?

Answer 9

Have to breed a litter of progeny then do brother - sister mating to get -/- mice.

Question 10

What are the benefits of mutagenesis screens?

Answer 10

You can create valuable mutations in tissues without having any specialist knowledge of those genes

You can make mutations that are impossible to make deliberately or would never have been thought

Question 11

What are the disadvantages of mutagenesis screens?

Answer 11

Uses very large numbers of animals

Wasteful

Question 12

What is meant by homologous recombination?

Answer 12

Occurs during meiosis

Identical homologous sequences on maternal and paternal chromosomes find each other, line up and may cross over.

Can be used to introduce DNA into new cells

Chiasmata is the term used to refer to the point at which the crossing over occurs

Question 13

How do you replace a gene by homologous recombination?

Answer 13

You want to replace a piece of functional DNA with a non-functional DNA (called a transgene or a targetting vector)

Give the transgene flanking DNA that is identical to the DNA surrounding the original strand of DNA.

Introduce this DNA into the cell and hope that homologous DNA sequences find each other and cross over

Question 14

What things might you add to the ‘non-functional DNA’ ?

Answer 14

Green flourescent protein

Neomycin resistance marker

These are important to allow you to select yout recombinant DNA

Question 15

How do we negatively select cells that have randomly taken up the DNA (without homologous recombination)?

Answer 15

The DNA strand contans a Thymidine Kinase gene beyond the flanking regions. This means that the TK gene only exists if it has been randomly introduced into a gene by DNA repair enymes. These cells can be killed using gangciclovir - leaving only the genes that have been introduced by homologous rcombination.

Question 16

After positive and negative selection, how do we test the integrity of the ntegrated vector?

Answer 16

PCR and southern blotting on genomic DNA

Question 17

The only surviving cells are:

Answer 17

The cells that have participated in the double homologous recombination

Question 18

This slide is essentially saying that this double homologous recombination event is very unlikely to take place on both copies of the chromosome in ES cells. Therefore when ES cells have been successfully targeted (i.e the gene has been replaced) the ES cells are implanted into the ICM of the host blastocyst, then they are implanted into the uterus of the pseudopregnant female. Offspring is chimeric.

From here we hope that the germline cells also contain this mutation. This species will be heterozygous - they need to breed with other heterozygous mice in order to create a mouse that is homozygous

Question 19

What are the potential set backs for those wanting to perform gene knockout?

Answer 19

Homologous recombination is a very rare event

Some cells that have passed through positive and negative selection contains DNA that is distorded from the original DNA

Very lengthy process

Question 20

What are the potential variations of knockout interpretations?

Answer 20

Redundancy can lead to mild or no phenotype at all. For example knockout of the MyoD gene has no phenotype because there are other functional genes that can fulfill the role of muscle production without the aid of MyoD

Alternatively Early embryonic lethality may occur. For example Oct 4 is a gene responsible for pluripotency

Some gene knockouts may affect how mice behave in the wild however in captivity you would never be able to examine this behaviour

Some genes act differently amongst different species

Genes don’t act in isolation - there may be many teps between gene dysfunction and animal phenotype