Design and Generation of Gene‐Targeting Vectors
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Abstract
This unit provides an overview of the major types of mutant alleles that can be generated by gene targeting in ES cells. It presents the growing public resources of premade gene targeting vectors, modified ES cells, and mutant mice. General guidelines for the design of targeting vectors are followed by protocols for the construction of vectors to generate knockout (KO), conditional KO, and subtle mutant alleles. Curr. Protoc. Mouse Biol. 1:199?211. © 2011 by John Wiley & Sons, Inc.
Keywords: gene targeting; conditional KO mice; targeting vector design; knockout mice; EUCOMM/KOMP
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- Introduction
- Protocols for the Construction of Gene Targeting Vectors
- Concluding Remarks
- Literature Cited
- Figures
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Figure 1. Mutant alleles generated by gene targeting and gene trapping. To generate a germline KO allele a neomycin‐cassette is inserted into the critical exon 2 of the target gene. This leads to the disruption of the gene's translational reading frame and the premature termination occurs at the neo ‐polyA site (A ). In a gene trap allele, a βgeo‐cassette, harboring a splice acceptor (SA), the β‐galactosidase/neo coding region and a polyA sequence, is inserted, resulting in the truncation of the wild‐type transcript (B ). Using gene targeting, a subtle mutation can be inserted into an exon of the target gene (C ). In a conditional knockout allele (D ), the critical exon 2 is flanked by two Cre recombinase recognition sites ( lox P sites). Upon Cre expression, the critical exon is excised, leading to a reading frame shift upon splicing to the downstream exon 3. The “knockout first, conditional‐ready” allele (E ), which is used by the EUCOMM/KOMP consortia, allows classical and conditional gene targeting (see Fig. 2). Exons of the target gene are shown as numbered rectangles. View Image -
Figure 2. Gene targeting using “knockout first, conditional‐ready” alleles. The “knockout first, conditional‐ready” allele (Tm1a, A ) consists of a β‐galactosidase reporter cassette (“ lacZ ‐pA”) and a neomycin expression cassette (“act‐ neo ‐pA”) flanked by two FRT sites. The critical exon 2 is flanked by two lox P sites; a third loxP site is located between the lacZ and neo genes. Upon Cre recombination of the Tm1a allele, exon 2, and the neomycin cassette are excised, leading to the lacZ ‐tagged knockout allele (Tm1b, B ). If the knockout first allele is initially recombined using FLP recombinase, the β‐galactosidase cassette is deleted, creating a conditional knockout allele (Tm1c, C ). Subsequent recombination with Cre recombinase in specific cell types leads to the conditional inactivated allele (Tm1d, D ). Exons of the target gene are shown as numbered rectangles. View Image -
Figure 3. Targeting vectors for germline knockout alleles. Genomic DNA or a BAC clone (A ) is used for the PCR amplification of homology arms, which are then cloned into the plasmid backbone (B ). The resulting targeting vector (C ) is integrated into the target gene locus by homologous recombination, leading to a neomycin‐positive knockout allele (D ). Exons of the target gene are shown as numbered rectangles. View Image -
Figure 4. Targeting vectors for conditional knockout alleles. Genomic DNA or a BAC clone (A ) is used for the PCR amplification of homology arms, which are then cloned into the pEasyFlox‐II‐DTA plasmid backbone (B ). The resulting targeting vector (C ) is used for the mutagenesis of the target gene by homologous recombination, leading to a neomycin‐positive mutant allele (D ). Finally, the neomycin cassette is excised using FLP recombinase to obtain the conditional allele (E ). Exons of the target gene are shown as numbered rectangles. View Image -
Figure 5. Targeting vectors for insertion of subtle mutations. Genomic DNA or a BAC clone (A ) is used for the PCR amplification of homology arms. A subtle mutation and a silent mutation creating the restriction site Re2 are introduced by the use of a PCR approach for site‐directed mutagenesis. The homology arms are then cloned into the plasmid backbone (B ). The resulting targeting vector (C ) is integrated into the target gene locus by homologous recombination, leading to a neomycin‐positive mutant allele (D ). Finally, the neomycin cassette is excised using FLP recombinase to obtain the mutant allele harboring the subtle mutation (E ). Exons of the target gene are shown as numbered rectangles. View Image
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