Conditional gene manipulation: Cre-ating a new biological era

Abstract

To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase (SSR) systems (Cre-loxP, Flp-FRT, and ΦC31) have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems (tetracycline and tamoxifen), stage-specific knockout and transgenic expression can be achieved. The application of this "SSR+inducible" conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.

Fig. 1

DNA manipulations controlled by Cre and loxP directions or locations (a) If the two loxPs are in the same direction on one DNA molecule, the DNA between them will be deleted. (b) When one loxP is on a linear DNA while another is on a circular DNA, the circular DNA will integrate into the linear DNA at the target. Small circular DNA is easily lost in cells; hence, the reaction in (a) happens more easily than that in (b). (c) If two loxPs are opposite, the DNA fragment between them will be inverted. (d) The DNA molecules will exchange a segment if both loxPs lie on linear DNA molecules. The black triangles are loxP sites, and indicate the direction. The length of the arrows indicates the relative tendency of reactions. Modified from Nagy (2000)

Fig. 2

Conditional gene knockout (a) In embryonic stem (ES) cells, the wild-type allele recombines with the targeting vectors on two homologous arms. This homologous recombination introduces two FRT sites flanking a selection marker neomycin-resistance gene (neo) and two loxP sites flanking the gene (or exon) of interest. (b) The Flp expression in the ES cells results in the excision of neo. (c) At the appropriate stage (usually in mice), the gene (or exon) is excised with the Cre expression. The rectangle (except TK) is the gene of interest (or exon). The black triangles indicate the loxP sites, and the black arrows indicate the FRT sites. TK: thymidine kinase gene. Modified from Guan et al. (2010)

Fig. 3

ΦC31 integrase system The ΦC31 integrase catalyzes recombination between the attP and attB sites to form the attL and attR sites. The attL and attR sites are the hybrid sites, which are no longer recognized by the ΦC31 integrase. Modified from Groth et al. (2000)

Fig. 4

Tetracycline-inducible system (a) Tet-Off system. tTA is active without Dox (or tetracycline), and the gene of interest is expressed. With Dox (or tetracycline) treatment, tTA is inactivated, and the gene is no longer expressed. (b) Tet-On system. rtTA is inactive without Dox (or tetracycline), and the gene of interest is not expressed. With Dox (or tetracycline) treatment, rtTA is activated, and the gene is expressed

Fig. 5

Tamoxifen-inducible system In the absence of tamoxifen, Cre-ER binds with Hsp90, and is located in the cytoplasm. When tamoxifen is present, it binds with the ER, displacing Hsp90. The Cre-ER-tamoxifen complex translocates to the nucleus, in which Cre executes its function. The black triangles indicate loxP sites. Modified from Tian et al. (2006)

Fig. 6

Stage-specific gene expression in transgenic animals The floxed STOP cassette is inserted between the promoter and the first exon of the gene. Transgenic animals may be generated using the construct, but exons following the STOP cassette are not transcribed. With the expression of Cre, the STOP cassette is cut out, restoring the normal expression of the gene. The black triangles indicate loxP sites. Modified from Dragatsis and Zeitlin (2001)

Fig. 7

Conditional gene trapping in the EUCOMM (a) Trapped allele. The trapping vector inserts into the genome of the ES cells. With a splice acceptor, a fusion transcript is formed between the upstream exons of the gene and the selection/reporter cassette. The transcription of the downstream exons is terminated, and results in the mutation of an endogenous gene. (b) Transient step for inverting the trapping vector with Flp recombinase. By expressing Flp recombinase in the ES cells, the selection/reporter cassette is inverted from a pair of F3 or FRT. Consequently, a pair of homotypic F3 or FRT sites is placed in the same orientation. Recombination between these two homotypic sites excises the other heterotypic site between them, and the selection/reporter cassette is locked against re-inversion. (c) Conditional allele. The inversion restores the gene function in the ES cells because the selection/reporter cassette is in the opposite orientation for trapping genes. (d, e) Transient step and trapped allele in specific tissues. By breeding the mouse from the ES cell line in (c) with a tissue-specific Cre mouse, the trapping vector is re-inverted, resulting in a conditional mutation in specific tissues depending on the Cre mouse line. The mechanism of re-inversion is the same as that of the inversion in the Flp-FRT/F3 system in (a) and (b). SA: splice acceptor; βgeo: a fusion of neo and β-gal; pA: poly A. Modified from Schnütgen (2006)

Fig. 8

Removable gene trap A floxed trapping cassette is inserted within a gene (between exon1 and exon2 in this case). The transcription is “trapped”, and only exon1 is translated, resulting in a non-functional protein. When Cre is expressed, the trapping cassette is excised, and the gene resumes its normal transcription and translation. The black triangles indicate loxP sites. Modified from Ishida and Leder (1999)

Fig. 9

Multipurpose conditional alleles The targeting vector contains the floxed gene (or exon) and a trapping cassette flanked by two FRT sites. After homologous recombination in ES cells, the gene is targeted. Gene expression is interrupted because of the trapping cassette. If the gene trap causes embryonic lethality, the ES cells can be transfected with Flp, and the trapping cassette is excised, resulting in a conditional allele. Alternatively, the ES cells may be transfected with Cre, and generate null alleles with the critical exon deleted as in the conventional knockout. Modified from Friedel et al. (2007)

Fig. 10

Introducing point mutations into genes Similar to gene knockout, the wild-type allele is recombined with the targeting vector with a designed point mutation and a floxed selection marker neo. The selection marker neo can be cut out by Cre, leaving the point mutation and one loxP site in the genome. The black triangles indicate loxP sites. Modified from Gao et al. (2004)

Fig. 11

Generation of large-scope deletions using the two vectors in the MICER The 5′Hprt and 3′Hprt vectors are used in two targeting steps to introduce the two loxP sites into the mouse genome. The 5′Hprt and 3′Hprt fragments are kept in the same orientation if a large-scale deletion is to be performed. The DNA between the two loxP sites is deleted by the excision of the Cre recombinase in the ES cells. After the excision, the 5′Hprt and 3′Hprt will form an intact Hprt gene, and positive ES cell clones can be selected by hypoxanthine, aminopterin, and thymidine (HAT). P: puromycin resistance gene; Ag: K14-Agouti transgene; N: neomycin resistance gene; Ty: tyrosinase minigene. The black triangles indicate loxP sites. Modified from Yu and Bradley (2001) and Klysik (2002)