Knockout and transgenic mice: uses and abuses

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

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

Knockout variations

Ablation of gene

Neo Exon 2

Exon 1 Exon 2

X

Homologous DNA

XPlasmid with target gene

DNA injected into plasmid

Exon 1 Exon 2

Exon 1 Exon 2

Mutation of gene Desired mutation

Plasmid with target gene

DNA injected into plasmid

XX

Homologous DNA

Then you take your plasmid…

Neo Exon 2

And inject it into an ES cell

Exon 1 Exon 2

X X

Plasmid

Genomic DNA in ES cell

Neo Exon 2 Genomic DNA

which won’t produce a functional gene

Creating knockout mice for fun and profitInjections to produce superovulation

X

X

Sterile male Pseudo-pregnant female

Two days after mating, harvest blastocysts and inject genetically targeted embryonic stem cells

Inject blastocysts into uterus

Chimeric malesES cells must enter their germline

X

50% wild-type, 50% heterozygous (+/-)

Breed hets

25% homozygous knockouts (-/-)

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

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)

Exon 1 Exon 2

X

Exon 2

LoxP sites and everything in between them is excised

Cre

Knockout only in promoter region

Cre

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

Injections to produce superovulation

X

X

Sterile male Pseudo-pregnant female

One day after mating, harvest 1-cell embryos and inject DNA construct

Creating transgenics

Inject embryos

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

Cool transgenic tricks

• Generalized overexpression• Reporter genes• Bicistronic reporters• Toxic genes• Dominant negatives• Targeted oncogenesis for

immortalized tissue cultures• Tetracycline-regulated expression

Reporter genes

L7-GFP

Purkinje cellsglow green

Use to identifyPurkinje targetsin brainstem

Sekirnjak et al., 2003

Bicistronic reporters

My Gene B-gal

IRES

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.

Promoter

CAP-independentCAP-dependent

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

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

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.

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

So what’s the catch?