Knockout and transgenic mice: uses and abuses
Dec 24, 2015
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?