Transgenic Mice


Transgenic mice carry exogenous genetic material introduced by the experimenter. Homologous recombination is used to introduce programmed modifications of the mouse genome.

Keywords: transgenic mice; targeted mutagenesis; embryonic stem cells; gene function; genetic alterations

Figure 1.

Generation of transgenic mice by DNA microinjection into the pronucleus of the zygote. A DNA solution is injected (1) into the pronucleus of a zygote. Injected eggs are then reimplanted into a foster mother (2, 3). In a proportion of cases, the injected DNA integrates into the chromosomes of the zygote. The integrated exogenous DNA (the transgene) is transmitted through cell division to all the cells of the mouse born from the injected zygote, giving rise to a transgenic mouse. The presence of the transgene in the host DNA is monitored by Southern blot, using a radioactive probe that specifically recognizes the injected DNA (4, 6). Crossing of transgenic founders (F0) with a nontransgenic mouse will give rise to F1 progeny, half of which are heterozygous for the transgene (5, 6). F1 intercrosses (7) allow the experimenter to obtain mice homozygous for the transgene, therefore generating a line of transgenic mice.

Figure 2.

Different stages in the creation of genetically modified mice using embryonic stem (ES) cells.

Figure 3.

Hybrid transgenes and their use. A hybrid transgene is made of the regulatory sequences of a gene A bound to the coding sequences of a gene B. In a mouse carrying such a transgene, a gene of interest may be expressed in the cells or tissues where gene A is normally expressed. The gene of interest could code for: (1) A reporter gene (e.g. β‐galactosidase or green fluorescent protein). This will help in determining precisely the pattern of expression of gene A. (2) Any protein. The phenotypic consequences of the ectopic expression of this protein will give insight into its function. This approach may also allow study of the function of gene products in various pathological situations, for example the role of oncogenes in malignancy. (3) A toxin, which will result in the ablation of a given type of cells and could illuminate the physiological function of the ablated cells. (4) An immortalizing oncogene, in which case cells expressing the transgene in the mouse may serve to derive cell lines in culture, from cell types that otherwise could not be maintained in vitro. (5) A protein of biological or medical interest. The hybrid transgene is constructed in such a way that the protein of interest will be synthesized in a tissue from which it could be extracted and purified, for example resulting in the extraction of milk or blood. Owing to its small size, the mouse serves only as a model system for bigger animals, for example farm animals.

Figure 4.

General principle of homologous recombination (HR): (a) Knockout. The targeting vector includes a selection cassette inserted in an exon (exon 2; black rectangles: coding region; white rectangles: noncoding regions) and surrounded by regions of homology with the target gene. In addition, a cassette may be added at one end of the targeting vector, in order to counterselect the cells in which the integration occurs outside of the targeted gene; a cassette expressing the A subunit of the diphtheria toxin (DT‐A) is shown. Upon random integration, the DT‐A‐expressing cassette is retained and the cell is killed by the toxin. In contrast, upon HR, it is excised and therefore the cell survives. Recombination with the endogenous gene occurs within the homologous sequences and results in the creation of a null allele in which disruption of the gene is induced by insertion of the selection cassette into an exon. (b) Knockin: Besides invalidation of the target gene, a gene of interest is introduced in the locus. Following homologous recombination, the gene of interest is placed under the control of the promoter and regulatory sequences of the target gene, and is therefore expressed in place of the target gene. cDNA: complementary deoxyribonucleic acid (DNA); STOP: termination codon.

Figure 5.

Subtle mutations. The persistence in a modified allele of a selection cassette with its own promoter and regulatory sequences may affect the target locus and surrounding loci. Creation of subtle mutations (point mutations, small deletions and insertions, etc.) therefore requires elimination of the selection cassette. The two strategies that may be used to create this type of modification are shown. Left: the double replacement strategy. This approach requires the use of embryonic stem cells (ES) bearing a null mutation in the endogenous hprt gene. The first step (a) consists of introducing a cassette expressing the hprt gene in the target gene. The recombinant cells (hprt+) are selected in the presence of hypoxanthine, aminopterin, thymidine (HAT). Homologous recombinants are identified using Southern blot. In the second step (b), targeted ES cells are transfected with a replacement vector presenting a subtle mutation (asterisk) and devoid of a selection cassette. The homologous recombination event results in the loss of the hprt expression cassette. Targeted cells therefore revert to an hprt− phenotype, an event selected in the presence of 6‐thioguanine (6‐TG) (in principle, all the hprt− clones could result only from HR). The use of other replacement vectors carrying different modifications permits the rapid creation of several alleles for the same target gene. Right: Use of the Cre–loxP system (see Figure ). In the first step (c), the target gene is modified by a target vector with a subtle mutation and a ‘floxed’ selection cassette, that is, surrounded by two loxP sites in the same orientation. Then (d) the transient expression of Cre recombinase in the recombinant cells induces deletion of the selection cassette. Apart from the desired subtle modification, only one loxP site of 34 bp persists in the final modified allele. The position of this loxP site is chosen such that it does not interfere with the expression of the target gene (generally in an intron).

Figure 6.

The Cre–loxP system and its applications. (a) The loxP site (triangle) is a sequence of 34 bp composed of palindromic sequences of 13 bp separated by a sequence of 8 bp. Cre recombinase specifically recognizes this sequence, provokes the cleavage in DNA (vertical arrows) and (b) induces the recombination of DNA between the two loxP sites. This reaction is reversible. Several types of recombination events can be produced depending on whether the two loxP sites are carried by the same DNA molecule (recombination in cis) or by two different DNA molecules (recombination in trans) and depending on the respective orientation of the two loxP sites (the orientation of a loxP is given by the nonpalindromic 8‐bp sequence). (c) Recombination in cis. If the two loxP sites have the same orientation, the DNA region situated between these sites is deleted during recombination. This type of configuration is used to create mutations devoid of the selection cassette (see Figure ), deletions and conditional mutations (see Figure ). If the orientation of the two loxP sites is opposed, recombination leads to the inversion of the region comprised between the two sites. (d) Recombination in trans. If one loxP site is integrated in the genome and the other is carried by a circular plasmid, there may be an insertion of sequences carried by the plasmid in the integrated loxP site. However, since the insertion is a rare event compared with deletion (i.e. the reverse reaction), this type of event requires the use of mutant loxP sites. When the loxP sites are both integrated in the genome, recombination in trans induces chromosomal rearrangements: deletions, duplications or translocations. Such recombination events are rare and have to be selected to be revealed. To do so, one can use truncated and nonfunctional hploxP and loxP‐rt selection cassettes. After recombination between the loxP sites, and only in this case, a functional hp‐loxP‐rt cassette (the remaining loxP site is situated in an intron) is reconstituted, thus allowing selection of the chromosomal rearrangement desired (see legend to Figure ). Furthermore, the relative orientation of loxP sites compared with the centromeric–telomeric axis of the chromosomes is important. Indeed, in the case of wrong relative orientation, recombination will result in the formation of acentric or dicentric chromosomes, which, in view of their great instability, will be eliminated and induce cell death.

Figure 7.

Conditional gene targeting. (a) Creation of a ‘floxed allele’ for conditional gene targeting. Step 1: the targeting construct contains three lox P sites in the same orientation, sites 1 and 2 flanking an essential region of the target gene (here an exon), and sites 2 and 3 flanking the neo selection cassette. Step 2: transient expression of the Cre recombinase in the targeted cell results in three types of alleles: (1) type I contains the deletion, the phenotype of which can be assessed in vivo after transferring the mutation back into the animal; (2) type II corresponds to the ‘floxed allele’. In vivo deletion can be obtained by crossing mice carrying this allele with Cre‐expressing transgenic mice; shown is a scenario that results in the disruption of the floxed gene, specifically in neurons; (3) the third allele can also be recovered but usually has no applications. (b) Conditional gene targeting is obtained by crossing two transgenic mice. The first one (mouse A) carries two ‘floxed’ alleles of a given gene (type II, see (a)) and exhibits no phenotype (only one allele is shown). The second one (mouse B) is a transgenic mouse for a hybrid transgene corresponding to the Cre recombinase coding sequence under the control of the cis‐acting regulatory elements of a tissue‐specific promoter (here a neuron‐specific gene, Pneuron). In the progeny, only neurons express the Cre recombinase and consequently harbor deleted (type I, see (a)) allele; all other cells retain the active ‘floxed’ allele. Consequences of the absence of the target gene in neurons can therefore be assessed. Function of the target gene in other cell types could be addressed by crossing the first mouse with another transgenic mouse expressing the Cre under the control of an appropriate promoter.



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Further Reading

Cid‐Arregui A and Garcia‐Carranca A (1998) Microinjection and Transgenesis – Strategies and Protocols. New York, NY: Springer‐Verlag.

Cohen‐Tannoudji M and Babinet C (1998) Beyond ‘Knock‐out’ mice: new perspectives for the programmed modification of the mammalian genome. Human Molecular Reproduction 4: 929–938.

Houdebine L‐M (1997) Transgenic Animals – Generation and Use. Harwood Academic Publishers.

Leighton PA, Mitchell KJ, Goodrich LV, et al. (2001) Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410: 174–179.

Lewandoski M (2001) Conditional control of gene expression in the mouse. Nature Reviews Genetics 2: 743–755.

Robertson EJ (1987) Teratocarcinomas and embryonic stem Cells: A Practical Approach. Washington, DC: IRL Press.

Rodriguez CI, Buchholz F, Galloway J, et al. (2000) High‐efficiency deleter mice show that FLPe is an alternative to Cre‐loxP. Nature Genetics 25: 139–140.

Special issue (1998) Stem cells and transgenesis. International Journal of Developmental Biology. 42.

Stanford WL, Cohn JB and Cordes SP (2001) Gene‐trap mutagenesis: past, present and beyond. Nature Reviews. Genetics 2: 756–768.

Web Links

Nagy Lab. Cre‐expressing mice. Samuel Lenenfeld Research Institute, Mount Sinai

TBASE (The Transgenic/Targeted Mutation Database). Knockout mice. The Jackson Laboratory

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Babinet, Charles(Jan 2006) Transgenic Mice. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0005582]