Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene. The ROSA26 locus is such a genomic site that is ideal to integrate a transgene of interest for stable expression. Creative Biolabs has profound expertise in the generation of Rosa26 site-specific gene knock-in models and is willing to share our expertise with our customers to facilitate their brilliant studies.
Introduction of the ROSA26 Locus
Located on mouse chromosome 6, the Gt(ROSA)26Sor (ROSA26) locus spans around 9 kb and consists of three exons. It is moderately expressed with levels varying between different tissues in pre- and post-natal life. Currently, ROSA26 is a safe harbor locus broadly used for both constitutive and conditional gene expression in mice. It is proven “safe” in mice because the transgene is adequately expressed without perturbing endogenous gene structure or function.
Targeting the ROSA26 Locus in Mouse ES Cells
Generally, there are three different types of ROSA26 locus knock-in. In the original design, the transgenic cDNA is expressed constitutively from the ROSA26 promoter. Specifically, a splice acceptor (SA) sequence followed by the cDNA of the transgene of interest is inserted into an Xba1 restriction site within the first intron of the ROSA26 gene. A stop cassette, composed of a neomycin resistance gene (NeoR, a positive selection marker) and three polyadenylation (pA) sites, is inserted upstream of the transgene. A variation of the first strategy for conditional transgene expression is realized by using a STOP cassette flanked by two loxP sites. In this case, the presence of STOP can prevent the transgene from expressing unless the Cre is expressed to remove the STOP sequence via homologous recombination.
However, the moderate strength of the ROSA26 promoter may in some instances not able to achieve the desired levels of transgene expression. To overcome this limitation, an exogenous promoter or an enhancer promoter (e.g., CAG promoter) is introduced to drive high transgene expression from the ROSA26 locus. Moreover, to monitor expression of the transgene in vivo, an IRES-GFP cassette flanked by frt sites can be cloned downstream of the transgene.
Features of ROSA26 Knock-in Models
ROSA26 locus targeting approach allows a high frequency of correctly insertion and stable expression of a single-copy transgene. When combined with the Cre-loxP system, temporal and cell-type specific control of transgene expression can be achieved. What's more, as a targeted gene knock-in strategy, the number of mice required is less compared with random transgenesis.
CRISPR/Cas9 Targeting Strategy
Besides ESC/HR-based gene targeting technique, CRISPR/Cas9 system is also widely used to generate mouse models with targeted knock-in cDNA fragments at ROSA26 locus. SgRNA components targeting the ROSA loci in the genome are designed and synthesized, followed by co-injection of the guide RNAs and Cas9 enzyme. Homologous recombination (HR) will enable the donor fragment which contains loxP (or frt) sites to integrate into the breaking locus.
Other knock-in models that you might be interested in including:
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