Genetic manipulation technologies 3

Question 1

Define a transgenic animal

Answer 1

An animal whos every cell contains a exogenous gene (transgene) which was introduced artificially but can be naturally inherited by it’s offspring

Question 2

What 3 features do eukaryotic genes require that means they can function (as a transgene)?

Answer 2

1. promotor to drive expression in the desired tissue
2. An open reading frame encoding the gene you wish to express
3. Sequences that ensure correct mRNA processing - eg polyadenylation signals

Question 3

What are the methods of introducing transgene DNA into cells?

Answer 3

Direct injection - into cells using a micropipette

Electroporation

Chemical transfection

Infection

Question 4

What is chemical transfection?

Answer 4

Incubate cells in culture medium containing DNA and a chemical that wraps the DNA up and is either endocytosed or that diffuses through the cell membrane

Question 5

Describe how direct infection of transgenes works

Answer 5

A solution of DNA (transgene) is injected into a fertilised egg

This works best if you inject the transgene into the male pronucleus after fertilisation of oocyte has occurred BUT the nuclei have not undergone fusion

Question 6

One of the main issues with introducing transgenic DNA to an animals cells is how the DNA is processed in the cell after injection.

What is meant by this?

Answer 6

Integration is usually random (see previous deck)

Transgenes are often integrated as head-tail concatemers (repeated sequence) which are pretty useless

Question 7

For what reasons might transgene expression not be as much/more than expected?

Answer 7

Weak promoter / Insufficient regulatory elements

Copy number

Position effects (site of integration)

Epigenetic modification

Genetic background (farm animals, hence Dolly)

Very big transgenes (>1 Mb), which put the promoter of the transgene in its normal chromosomal context, usually work best