Pronuclear injection

Question 1

What is pronuclear injection?

Answer 1

Germline modification of the whole animal so that the modification is inherited
Developed in the 1980s in lab mice
A recombinant plasmid containing DNA injected into the pronucleus

Question 2

Describe the procedure

Answer 2

Stud male and super ovulating female that has been injected with reproductive hormones to release many eggs mate

The fertilised eggs are isolated

Microinject eggs with transgene DNA

Implant eggs into a pseudopregnant foster female that has been allowed to mate with a sterilised male

After 19 days the G0 pups are born

After 3 weeks isolate tail DNA for PCR and analyse by electrophoresis

Question 3

What is in the DNA that is injected?

Answer 3

Promoter
Intron
Polyadenylation sites

Question 4

What makes the promoter special?

Answer 4

Can be tailored to be constitutive (strong constant expression), tissue-specific, and highly regulated

Question 5

What makes the intron special?

Answer 5

Can be artificial

Contains binding sites and transcription factors for transgene expression

Question 6

What makes the polyadenylation sites special?

Answer 6

Allows polyA tail to be attached to mRNA needed for export of mRNA from the nucleus and translation

Question 7

Describe the first visibly transgenic mouse

Answer 7

1982
Eggs microinjected with metallothionein GH
Transcription induced by feeding zinc which is a regulated promoter
Transgene was expressed in multiple tissues
Increased GH protein in blood
Bigger

Question 8

Name some problems with PI

Answer 8

Can only be used to add genes not modify
Insertion in random locations (ectopic) and multiple copies
Sometimes get silencing of genes
Not always consistently inherited