Knockout and transgenic mice: uses and abuses

1. Knockout and transgenic mice: uses and abuses

2. Purpose: replace normal gene 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: insert new genetic material 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 Knockout mice Transgenic mice

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. Knockouts: first steps Ablation of gene Homologous DNA DNA injected into plasmid Neo Exon 2 X X Plasmid with target gene Exon 1 Exon 2 Mutation of gene Desired mutation Homologous DNA DNA injected into plasmid Exon 1 Exon 2 X X Plasmid with target gene Exon 1 Exon 2

5. Then you take your plasmid… And inject it into an ES cell Plasmid Neo Exon 2 X X Genomic DNA in ES cell Exon 1 Exon 2 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 Pseudo-pregnant female Sterile male

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

8. Cool knockout tricks • Knock-ins: replacement of endogenous gene with a different one, for example CaMKII T305 animals, for constitutively active or inactive proteins D (aspartic acid) acts as though it’s permanently phosphorylated A (alanine) prevents any phosphorylation

9. Cool knockout tricks 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 Cre Transgenic mouse Tissue-specific promoter 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 Injections to produce superovulation One day after mating, harvest 1-cell embryos and inject DNA construct X X Pseudo-pregnant female Sterile male Inject embryos

12. 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 • Reporter genes (where are my neurons?) • Bicistronic reporters (where is my gene?) • Toxic genes (what if we get rid of this neuron?) • Dominant negatives (why does my knockout suck?) • Tetracycline-regulated expression (how can we control the onset and offset of transcription?) • Targeted oncogenesis for immortalized tissue cultures (how can we grow these cells forever?) • Generalized overexpression (what does this gene do?)

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-independent CAP-dependent 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? Those nice knock-ins with the mutated phosphorylation site turn out to affect not just CaMKII inhibitory phosphorylation but also regulation of quantities of CaMKII…

21. 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), often in unexpected ways • Transgenes can disrupt endogenous genes by landing in the middle of them • Many labs are moving in the direction of temporally and spatially regulating genes in mutant mice, or using viral transfection, for tighter control of the results.