1. Knockout and transgenic mice: uses and abuses

2. Knockout mice Transgenic mice
To create: homologous integration of DNA in embryonic stem cells
Inserted DNA replaces normal gene at normal site on chromosome
Usually homozygote for best expression
Purpose: replace normal gene
To create: injection of new DNA into fertilized egg
Gene integrates randomly
Multiple copies in tandem
Expression level affected by integration site; may interrupt a gene
Purpose: insert new genetic material

3. Basic steps—knockout Pick a gene of interest
Knock it out or mutate it?
Create replacement construct
Inject into plasmid to cross over with gene of interest
Inject plasmid into stem cell and hope for recombination
Inject stem cell into blastocoel
Inject blastocyst into uterus

4. Knockout variations X X X X Ablation of gene Mutation of gene
Homologous DNA
DNA injected into plasmid
Neo
Exon 2 X X
Exon 1
Exon 2
Plasmid with target gene
Mutation of gene
Desired mutation
Homologous DNA
Exon 1
Exon 2
DNA injected into plasmid X X
Plasmid with target gene
Exon 1
Exon 2

5. Then you take your plasmid…
And inject it into an ES cell
Neo
Exon 2
Plasmid X X
Exon 1
Exon 2
Genomic DNA in ES cell
Genomic DNA
Neo
Exon 2
which won’t produce a functional gene

6. Creating knockout mice for fun and profit
Injections to produce superovulation
Two days after mating, harvest blastocysts and inject genetically targeted embryonic stem cells X X
Inject blastocysts into uterus
Sterile male
Pseudo-pregnant female

7. ES cells must enter their germline
Chimeric males
ES cells must enter their germline X
50% wild-type, 50% heterozygous (+/-)
Breed hets
25% homozygous knockouts (-/-)

8. Cool knockout tricks Tissue specificity with Cre-LoxP system
Knock-ins: replacement of endogenous gene with a different one, for example CaMKII T305 animals, for constitutively active or inactive proteins

9. Tissue-specific knockouts
Cre-Lox system
Cre recombinase snips out DNA between LoxP sites
A tissue-specific promoter in front of Cre produces tissue-specific snipping
Tissue-specific promoter
Cre
Transgenic mouse
e.g. L7 (Purkinje)
CaMKII (forebrain)
Cre X
Cre
LoxP sites and everything in between them is excised
Exon 1
Exon 2
Knockout only in promoter region
Exon 2

10. Basic steps—transgenic
Mutate or create a gene or fragment
Choose temporal regulation or not
Inject DNA construct into the male pronucleus of 1-cell embryos; hope for random insertion
Implant injected embryos into fallopian tubes

11. Creating transgenics X X Injections to produce superovulation
One day after mating, harvest 1-cell embryos and inject DNA construct X X
Sterile male
Pseudo-pregnant female
Inject embryos

12. Several transgenic lines stemming from different F1
Many offspring will carry the inserted DNA; only some will express it usefully X
Several transgenic lines stemming from different F1
Breed selectively
Usually both +/+ and +/- show expression

13. Cool transgenic tricks
Generalized overexpression
Reporter genes
Bicistronic reporters
Toxic genes
Dominant negatives
Targeted oncogenesis for immortalized tissue cultures
Tetracycline-regulated expression

14. Reporter genes L7-GFP Purkinje cells glow green Use to identify
Purkinje targets
in brainstem
Sekirnjak et al., 2003

15. Bicistronic reporters
IRES
Promoter
My Gene
B-gal
CAP-dependent
CAP-independent
IRES: internal ribosomal entry site
Both genes are expressed from the same mRNA, so you can tell when and where your transgene has been expressed
CAP is a sequence added in nucleus; normally it’s required for translation, but the IRES makes the second mRNA CAP-independent.

16. Dominant negative transgenes
Aim: to block protein kinase C (PKC) in Purkinje cells
Problem: PKC has several isoforms, so knockouts aren’t effective
Solution: PKCi transgene, which interferes with the regulatory portion of all PKCs, expressed under L7 promoter
De Zeeuw et al., 1998

17. Dominant negative transgenes
Aim: to block BDNF signalling through TrkB
Problem: BDNF can activate another receptor as well (p75)
Solution: TrkB-Tc transgene, which allows BDNF binding but prevents signalling
Saarelainen et al., 2003

18. Tetracycline regulation
Aim: to avoid developmental effects of transgene expression
Solution: Tet system, where a transgenic producing tTA is crossed with a transgenic with the tet-O promoter. tTA normally permits tet-O transcription, but in the presence of doxycycline it can’t.

19. So what’s the catch?

20. So what’s the catch?
Difficult to knock out genes in certain chromosome regions, near centromere
Knockout animals are often homozygous lethal
Alternatively, KOs/Tgs may show no phenotype at all
Lack of temporal or spatial specificity may perturb development and other brain regions
Compensation by upregulation of other genes (e.g. PKC)
Transgenes can disrupt endogenous genes by landing in the middle of them