Pronuclear injection Flashcards Preview

BIO2030 Biotechnology > Pronuclear injection > Flashcards

Flashcards in Pronuclear injection Deck (8)
Loading flashcards...

What is pronuclear injection?

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


Describe the procedure

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


What is in the DNA that is injected?

Polyadenylation sites


What makes the promoter special?

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


What makes the intron special?

Can be artificial
Contains binding sites and transcription factors for transgene expression


What makes the polyadenylation sites special?

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


Describe the first visibly transgenic mouse

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


Name some problems with PI

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