Transgenic, knockin and knockout mice Flashcards
What do we consider to be a transgenic mouse?
A mouse that has a piece of DNA introduced into it, regardless of origin
What is a transgenic mouse?
A mouse with a random insertion of a piece of DNA
What is a knockout mouse?
Targeted removal of sequences
What is a knockin mouse?
Targeted insertion of a transgene
What is on the vector is used to create a transgenic mouse?
Sequence of interest, strong or inducible mouse promotor, enhancer sequence, bacterial ori, bacterial selectable marker, MCS
Why would we use transgenic mice?
Overexpression of a WT allele, expression of a dominant negative mutant allele, expression of fusion proteins
How is a transgene typically inserted into the genome of a transgenic mouse?
As a head to tail array
How do you create a transgenic mouse?
- Remove an early embryo from a donor parent with coat colour 1, then extract ESC from the blastocyst.
- Transform the ICM cells and grow them in culture on selective media.
- Inject the transformed cells into a donor blastocyst with coat colour 2 and implant it back into the uterus of the donor mother
- Mate the chimeric mice to normal coat colour 2 mice to generate heterozygous progeny with coat colour 1
- Cross the heterozygote sibs to get the 1:2:1 ratio, where the homozygous transgenics have a slightly different coat colour than the heterozygotes
What is on the vector if we are trying to create a knockout mouse?
Homology arms, and in between the homology arms we have any exons we want to remain intact and a neomycin resistance cassette flanked by Frt sites. Outside the homology arms is the TK ORF
Why is it super important that the TK ORF is outside the homology arms?
If homologous recombination happens, then TK is not in the genome. But if non homologous recombination occurs, then TK is in the genome and turns gangcyclovir into a toxic product
What media would the transformed knockout cells be on?
Media with neomycin and gangcyclovir
How do we get rid of the selectable marker in a knockout or knockin mouse if it’s causing problems?
Cross the knockout mouse to a mouse expressing flippase, which will remove the neomycin cassette between the Frt sites
What is a conditional knockout?
The knockout only happens under certain conditions
What is the targeting vector for a conditional knockout mouse if we are trying to have a conditional knockout of exon 2?
Homology arms, and in between 2 loxP sites there is exon 2 and the neomycin resistance cassette flanked by Frt sites, which is in an intron
How do we do a conditional knockout?
The knockout only happens if Cre recombinase is expressed. So we can cross the conditional knockouts to mice expressing Cre recombinase in a certain tissue so YFG is only knocked out in that tissue but stays intact otherwise
