Gene targeting in mice: a review

Abstract

The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (1) delete or insert mutations into genes of interest to determine their function, (2) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g., mice susceptible to infections by human-specific pathogens of interest, (3) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (4) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse's lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice.

Figure 1A. Summarized most common steps for isolation of mouse embryonic stem (ES) cells and generation of homologous recombinant ES clones

(1) In the first step 3.5-day-old mouse embryos (blastocysts) are collected from the uterine horn of superovulated (hormone treated) mated female mice with, for example, an agouti coat (strain 129/Sv). (2) Embryonic stem (ES) cells (ESCs) are derived from the inner cell mass of blastocysts and cultured on a feeder layer of mitotically inactivated mouse embryonic fibroblasts (MEFs), in ESC medium (supplemented with leukemia inhibitory factor (LIF). (3) After electroporation with the targeting vector of interest, (4) successfully transfected ESCs are selected by adding appropriate selection agent to the ESC medium; and (5) ESC clones are picked. (6) Homologous recombinant ESC clones are identified by Southern blot. The genomic DNA isolated from ESC clones should be digested with an appropriate restriction enzyme that produce one cut inside the targeting vector and one cut just outside (upstream or downstream) the targeting vector, in the targeted chromosomal region. The use of an “external” probe outside of the targeting construct will produce a band with a size corresponding to unmodified wild-type allele(s), which is here indicated by X kb, and, if homologous recombination occurred, a second band of bigger or smaller size corresponding to the targeted allele, which is here indicated by X-Y kb.

Figure 1B. Generation of mice with genome modification of interest using homologous recombinant ESC clones

(1) If ESCs are derived from mice with an agouti coat (such as strain 129/Sv), the recipient pre-implantation mouse embryos (blastocysts) should be collected from female mice with black coat (such as strain C57BL/6). (2) The identified and expanded homologous recombinant ESC clones (see Figure 1A) are injected into recipient pre-implantation mouse embryos (blastocysts) that are collected from female mice with black coat (strain C57BL/6).(3) These injected blastocysts are then surgically transferred to a recipient pseudopregnant foster mother to allow the embryos to develop. Females of CD1 mouse strain make very good mothers, and are thus used by several laboratories as foster mothers. (4) Because ESCs and recipient blastocysts were derived from mouse strains with distinguishable coat-colors, the desired chimeric offspring can be visually recognized by inspection of coat-colour chimerism (% of black and agouti hair on the mouse black-agouti). (5) Chimeric offspring (usually only the males, because the used ES cell lines are usually male) are mated with C57BL/6 mice to produce the F1 generation. (6) The germline transmission is then confirmed by Southern blot analysis or PCR of tail DNA from the agouti (not black) mice of the F1 generation.

Figure 2. Typical gene targeting strategy

A targeting vector is typically composed of three basic units: (i) a 5’ homology arm; (ii) a gene marker for positive selection (e.g. neomycin resistance gene (neo)); (iii) a 3’ homology arm; and (iv) a negative selection marker (neg. sel. Marker), such as thymidine kinase, diphtheria toxin fragment A (DT-A), or, if the positive selection marker is flanked by loxP sites, Cre recombinase gene (Cre). Furthermore, any desired DNA sequence of interest (here green box) can be inserted between the homology arms of the targeting vector, in order to introduce it into the target genome by homologous recombination. Homologous recombination between the targeting vector and the target cognate chromosomal region results in the disruption of one genomic copy of the targeted genomic locus and loss of the vector’s negative selection marker gene. Crossover points are depicted by “X”. The floxed (loxP sites flanked) positive selection marker gene can be removed by expressing Cre recombinase in the recombinant ESCs or by crossing the chimeric mice with Cre-expressing transgenic mice (see also Fig. 4A).

Figure 3. LoxP structure and Cre recombinase-mediated recombinations

(A) Single loxP site that contains two inverted 13 bp repeats, separated by an asymmetric 8 bp long sequence. The type of Cre-mediated recombination is dependent on the orientation and location of the loxP sites: (B) Cre excises a circular molecule from between two loxP sites placed in the same orientation; (C) Cre inverts the DNA sequence between two loxP sites positioned in opposite orientation; (D) Cre-mediated recombination between two different linear DNA molecules (e.g. chromosomes), each containing a loxP site, resulting in the exchange of the DNA regions flanking the loxP sites. Figure was modified from Torres and Kuehn (111).

Figure 4. Conditional gene targeting using the Cre/loxP recombination system

(A) Cre-mediated inactivation of a gene of interest. Left mouse: introduction of loxP sites into a genomic locus of interest by homologous recombination using embryonic stem cells (see also Fig. 1). LoxP sites are introduced in a manner that they don’t interfere with the function of the targeted gene. Right mouse: a transgenic strain that express Cre-recombinase under the control of cell type- or tissue-specific promoter. By crossing the floxed mouse with Cre transgenic mice, Cre mediates the deletion of the floxed genomic sequence, resulting in the inactivation of the targeted gene. Gene deletion is restricted to the “area” (cell types or tissues) where Cre is expressed (white flecks). (B) Cre-mediated activation of a gene of interest. Left mouse: introduction of a floxed intervening sequence (e.g. polyadenylation signal sequences) that prevent the correct transcription of the targeted gene. By crossing the floxed mouse with Cre transgenic mice, Cre mediates the deletion of the floxed intervening sequence, resulting in the reactivation of the targeted gene. Gene activation is restricted to the “area” (cell types or tissues) where Cre is expressed (white flecks).

Figure 5. Targeting transgenes into the ROSA26 locus

Introduction of gene cassettes of interest into Roas26 locus by homologous recombination in embryonic stem cells. A floxed intervening sequence, neomycin (Neo)-polyadenylation signal sequences (pA), prevents the transcription of the transgene. Cre expression mediates then the deletion of the floxed intervening sequence, resulting in the expression of the transgene and a reporter gene. The reporter gene facilitates to track the expression of the transgene. Numbered (1–3) blue rectangles: exons of the Roas26 locus. SA: splice acceptor. DNA-elements are not drawn to scale.