Flashcards in Pronuclear injection Deck (8)
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?
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