Overview: generation of gene knockout mice

Abstract

The technique of gene targeting allows for the introduction of engineered genetic mutations into a mouse at a determined genomic locus. The process of generating mouse models with targeted mutations was developed through both the discovery of homologous recombination and the isolation of murine embryonic stem cells (ES cells). Homologous recombination is a DNA repair mechanism that is employed in gene targeting to insert a designed mutation into the homologous genetic locus. Targeted homologous recombination can be performed in murine ES cells through electroporation of a targeting construct. These ES cells are totipotent and, when injected into a mouse blastocyst, they can differentiate into all cell types of a chimeric mouse. A chimeric mouse harboring cells derived from the targeted ES cell clone can then generate a whole mouse containing the desired targeted mutation. The initial step for the generation of a mouse with a targeted mutation is the construction of an efficient targeting vector that will be introduced into the ES cells.

Figure 1

A schematic of a replacement vector: Two homology arms flank a positive drug selection marker (neo^r). A negative selection marker (HSV-tk) is placed adjacent to one of the targeting arms. A unique restriction enzyme site is located between the vector backbone and the homology arm. When linearized for gene targeting, the vector backbone will then protect the HSV-tk from nucleases.

Figure 2

Gene inactivation through a replacement vector: Homologous recombination with a replacement vector requires a positive selection marker (neo^r), a negative selection marker (HSV-tk), and two targeting arms (homologous sequence is depicted with exons in light grey). A representative target gene (with exons in dark grey) is aligned with the targeting vector. For this example, the targeting vector is designed so that Exon 2 is substituted with the neo^r gene. The replacement of Exon 2 by the neo^r gene is then recapitulated in the target locus as homologous recombination exchanges genomic sequence for the homologous sequence of the targeting vector. Two homologous recombination events (depicted through the crosses) occur via the long and short targeting arms to introduce non-homologous sequence (i.e., neo^r) into the designated gene. Insertion of the neo^r gene is selected for by treatment of cells with neomycin sulfate (G418) in tissue culture. The negative selection marker (HSV-tk) is not recombined into the chromosome and is lost during gene targeting. If the targeting construct is randomly integrated anywhere in the genome, the HSV-tk gene would be intact. Random integrants can be selected against through either gancyclovir or FIAU treatment.

Figure 3

Gene targeting to insert a subtle mutation: In the targeting vector, a subtle mutation (depicted as ‘*’) is designed into one of the homology arms. Subtle mutations can consist of point mutations, micro-deletions, or insertions. When sequence in the target gene is exchanged with homologous sequence from the targeting vector, the subtle mutation is introduced into the chromosome. Homologous recombination occurs through both the long and short homology arms. A positive drug selection marker (neo^r) is needed in this strategy to select for clones that have undergone recombination. With a clean mutation, the neo^r gene is inserted into the targeted locus, but no exons are lost as a result of recombination. The neo^r gene needs to be removed so that it does not interfere with transcription of the recombined allele. LoxP sites (depicted as black triangles) are used to flank the neo^r gene in order to facilitate its removal. These 34 bp loxP sites are recognized by the Cre recombinase, which can be introduced into targeted stem cells by transient transfection. If the loxP sites are in the same direction, the Cre recombinase will circularize out any intervening sequence. With Cre-mediated recombination, only a single loxP site will remain. The loxP site in the recombined allele is situated within intron sequence so that it does not interfere with transcription of the mutant protein.

Figure 4

Gene targeting to create a conditional knockout: In this strategy, the targeting construct is designed so that a loxP site (indicated with a black triangle) is located next to an essential exon within the homology arm. A floxed neo^r gene is then positioned on the opposite side of this exon. Both the loxP site and the floxed neo^r gene are introduced into the target locus through homologous recombination. Unlike a conventional knockout experiment, the targeting vector is assembled so that no exons are lost as a result of homologous recombination. After the stem cell clone with the properly targeted insertion is identified, the Cre recombinase is then introduced into the cells through a transient transfection. The Cre recombinase can produce three types of recombinations. In the first example (shown as ①), both Exon 2 and the neo^r gene are excised from the chromosome leaving only 1 loxP site. In the second scenario (shown as ②), only Exon 2 is removed while the floxed neo^r gene remains in the targeted chromosome. In the third case (shown as ③), just the neo^r gene is excised while 2 loxP sites remain to flank Exon 2, creating a floxed allele. Only the stem cell clones with this specific recombination are injected into blastocysts to generate floxed mice. Conditional deletion of Exon 2 in vivo is then accomplished typically by breeding the floxed mice with a Cre-expressing transgenic line. After the removal of neo^r in tissue culture and later the conditional deletion of Exon2 in vivo, the final allele has only one loxP site remaining within intron sequence of the targeted gene (as depicted in ①).

Figure 5

Knock-in of a cDNA through the use of gene targeting: Knock-in constructs are similar in design to conventional knockout gene targeting vectors, except that additional sequence (i.e., a cDNA or protein domain of interest) is inserted into the target gene. Through homologous recombination, this foreign sequence is introduced in frame into the target locus to be expressed by its promoter. Essential coding sequence in the target locus is simultaneously lost during recombination with the targeting construct. In this example, homologous recombination places a cDNA under the control of the promoter of the target gene. Concurrently, the translational start sequence in Exon 1 of the target gene is also replaced by the cDNA. The cDNA, in this example, has a poly A addition signal (pA) which will stop any further transcription downstream of the targeted insertion. For a knock-in targeting vector, one of the homology arms must consist of genomic sequence upstream of the planned insertion site for the cDNA. To knock in a cDNA, as shown, a targeting vector must use promoter sequence for one of its homology arms (as depicted with the directional arrow). A positive drug selection marker (neo^r) is still needed to select for clones that have inserted the designated cDNA into the target gene. Two homologous recombination events serve to insert the cDNA and neo^r gene into the target location while knocking out Exon 1.

Figure 6

Gene inactivation with an insertion vector: For an insertion vector, only one homologous recombination event is needed for targeted insertion of DNA into a designated gene. In this case, recombination occurs around a double strand break that is located in the homology arm of the targeting construct. The entire insertion vector is then incorporated into the gene, including the plasmid backbone (represented by the thin line). A drug selection marker like the neo^r gene is still needed for positive selection, but this marker can be positioned either in the targeting arm or in the plasmid backbone of the insertion vector. In this example, the positive drug selection marker is designed in the homology arm in order to replace essential coding sequence of the target gene (as shown with the disruption of Exon 2 by the neo^r gene). For insertion vectors, a knockout allele is essentially generated because the target gene is disrupted with insertion of the neo^r gene and by duplication of exonic sequence.