Contemporary approaches for modifying the mouse genome

Abstract

The mouse is a premiere experimental organism that has contributed significantly to our understanding of vertebrate biology. Manipulation of the mouse genome via embryonic stem (ES) cell technology makes it possible to engineer an almost limitless repertoire of mutations to model human disease and assess gene function. In this review we outline recent advances in mouse experimental genetics and provide a "how-to" guide for those people wishing to access this technology. We also discuss new technologies, such as transposon-mediated mutagenesis, and resources of targeting vectors and ES cells, which are likely to dramatically accelerate the pace with which we can assess gene function in vivo, and the progress of forward and reverse genetic screens in mice.

Fig. 1.

Mutant mice can be generated either by microinjection or by tetraploid complementation. Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts and grown in culture. These cells can be modified by gene-targeting and then either microinjected into a host blastocyst to generate chimeras that are then bred for germline transmission (left) or alternatively used to make entirely ES cell-derived mice using tetraploid complementation (right). Usually the ES cells used are derived from a 129 mouse strain (or hybrids with a 129 strain) and carry the dominantly active agouti locus allowing coat color (brown) to be used for tracking both the contribution of ES cells to the formation of a chimera and for the identification of germline ES cell-derived offspring.

Fig. 2.

There are various designs of targeting vector that can be used to generate loss- or gain-of-function alleles in ES cells. The structure of a generic gene is shown with numbered boxes representing the exons (either coding, shown in gray, or noncoding, shown in white). Homologous recombination (shown by crosses) results in different modifications of the targeted locus depending on the nature of the targeting vector used. A: when using a standard replacement vector, typically a drug resistance marker (PGK-Drug^r) is inserted into the gene to disrupt a critical exon. B: knock-in vectors can be used to target expression markers such as LacZ into a locus. These vectors typically result in fusion of LacZ with an exon of the gene. With this approach the gene is disrupted and its expression tagged. C: elegant strategies can be used to generate both null and conditional alleles of a gene using the Cre/LoxP and Flp/FRT recombinase systems. LoxP sites are shown as red triangles, and FRT sites are shown as green triangles. Vectors of this design can be used to inactivate a gene somatically or in a particular cell or tissue type. D: “knockout first” alleles combine the features of both knock-in and conditional alleles to generate targeting events that result in null alleles that are tagged with LacZ to monitor gene expression. Endogenous transcripts of a gene are captured by the splice acceptor (SA) and terminated. These alleles can be treated with Flp to generate a conditional allele and then with Cre to generate a loss-of-function allele in a cell- or tissue-specific manner. E: insertion vectors, unlike replacement vectors, have a single length of homology and are linearized by cutting within the homologous sequence. When they target the genome they insert vector sequences such as the drug resistance marker at the insertion site. F: gene-trap vectors have a very similar design to knock-in vectors except that they are randomly introduced into the genome as opposed to being targeted to a specific locus. They usually contain SA sequences and fusions between drug resistance and expression markers such as neomycin and LacZ. G: it is also possible to generate gain-of-function alleles in mice. One common approach is to target a cDNA of interest behind a loxP-flanked STOP element, which prevents expression of the cDNA. Expression of the cDNA occurs after the STOP element is removed by Cre-mediated excision (41). Another strategy involves placing the cDNA of interest between inverted mutant loxP sites, shown as blue and purple triangles (62). After expression of Cre, the cDNA “flips” into the “on” position and is expressed. Importantly, recombination of these mutant loxP sites generates a variant loxP site that is unable to recombine again, locking the cDNA in the on position.

Fig. 3.

Recombineering is a powerful strategy for rapidly generating targeting vectors. Typically, bacterial artificial chromosomes (BACs) are modified using λ Red-mediated recombination in bacteria. Firstly a LoxP-flanked bacterial selection marker that carries kanamycin resistance (Kan^r) is targeted onto the BAC. This marker is then “popped out” by expressing Cre recombinase in the bacteria, leaving behind a single loxP site. The BAC is then retargeted with a second cassette carrying a neomycin-kanamycin (Neo/Kan^r) selection maker flanked by FRT sites and a single LoxP site. This vector carries a mammalian promoter such as phosphoglycerate kinase (PGK) and a bacterial promoter such as EM7, allowing drug resistance in ES cells and Escherichia coli, respectively. Finally, the modified fragment of DNA is “captured” from the BAC by a process called “gap-repair.” The backbone of the capture vector may contain a negative selectable marker such as diphtheria toxin (DTA), which suppresses random integration of the targeting vector in the genome. The resulting vector is a conditional allele.

Fig. 4.

Cre recombinase can be used to activate or delete a gene in a tissue-specific manner. Tissue-specific gene recombination can be performed either by 1) breeding the mouse carrying the conditional allele with a mouse expressing a tissue-specific Cre (i.e., whereby Cre is only expressed in a specific tissue type, such as liver represented as a blue circle) or 2) somatically by breeding the mouse carrying the conditional allele with a mouse expressing an inducible tissue-specific Cre (i.e., whereby tissue-specific expression of the Cre only occurs after induction of the promoter via treatment with a chemical, such as tamoxifen). In both cases, expression of Cre or a fusion between Cre and a truncated estrogen receptor sequence is driven by a tissue-specific promoter (e.g., Albumin-Cre, which switches on Cre expression only in the liver).

Fig. 5.

Gene targeting in ES cells to engineer chromosomes. A: insertional targeting vectors, as shown, can be used to insert loxP sites (red triangles), positive selectable markers (such as the neomycin resistance cassette, Neo, and the puromycin resistance cassette, Puro) and HPRT gene fragments [5′ (exons 1–2) and 3′ (exons 3–9)] into the mouse genome for chromosome engineering. These vectors are called the 5′HPRT and 3′HPRT vectors, respectively. After the expression of Cre recombinase recombination between these vectors results in the formation of a functional HPRT mini-gene and allows ES cells to be grown in HAT (hypoxanthine, aminopterin, and thymidine)-containing medium. HAT resistance allows in vitro selection of ES cells carrying the desired chromosomal rearrangement (3, 85). B: the Mutagenic Insertion and Chromosome Engineering Resource (MICER) library is composed of 5′HPRT and 3′HPRT vectors. i: The orientation of the genomic insert in these vectors will dictate the orientation of the vector when it is targeted to the genome. These orientations are color coded to facilitate the selection of MICER vectors required to generate the desired rearrangement. ii: Shown is an example of the MICER vector selection required to generate a deletion by targeting the vectors in cis-. iii: Over 100,000 of these 5′HPRT and 3′HPRT vectors have been end-sequenced and are displayed on the Ensembl genome browser. Further details on MICER clone configurations to generate different types of rearrangements has been published previously (83).

Fig. 6.

The bipartite transposon system is composed of a transposon and transposase. Transposons may function by a “cut-and-paste” mechanism to insert sequence (yellow box) between the transposon repeats (red arrows) into the genome. This “jumping” is catalyzed by the transposase enzyme (blue circles). Transposons, like all insertional mutagens, have preferences for the sequence into which they like to insert.