Many scientists have researched into gene targeting in mice that have made it possible to develop a number of tools for knockin genes in and out of the mouse genome. But there are still some genomic sites which are enable to add genes, since many are prone to silence or disrupt normal gene functions. The discovery of the Rosa26 locus and the hypoxanthine phosphoribosyltransferase (HPRT) locus overcomes these problems.Rosa26 and HPRT are known to be permissive for transgene expression, and are therefore ideal candidates to integrate a transgene to have stable and ubiquitous expression. Creative Animodel provides knockin mice and rat models which the transgene is inserted into HPRT locus and Rosa26 locus safely and targetedly.
The Rosa26 is a genomic mouse locus commonly used to knockin cDNA constructs for ubiquitous or conditional gene expression in transgenic mice and rat, it was originally identified in a gene-trap screen in murine embryonic stem (ES) cells, and was subsequently shown to be expressed during embryonic development and in adult mice. The locus is believed to encode three non-coding transcripts of unknown functions. The Rosa26 locus is commonly used in mice to achieve targeted insertions by homology-directed repair using ES cells and it has been targeted using sequence-specific nucleases in mice. The rat Rosa26 locus is targeted using ES cells but not yet using nucleases.
The X-chromosomal gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), first recognized through its human germinal mutations, quickly became a useful target for studies of somatic mutations in vitro and in vivo in humans and animals.The HPRT locus is often chosen as a “permissive” locus for targeted integration of transgenes in mouse ES cells. HPRT has the distinct advantage to support efficient selection for the anticipated homologous recombination event using hypoxanthine-aminopterin-thymidine media, wherein cell survival is dependent upon HPRT activity. When ES cells bearing a partially deleted and null HPRT locus are transfected with a targeting construct containing the deleted HPRT sequences and the experimental transgene, homologous recombination at the HPRT locus simultaneously restores functional HPRT expression and inserts a single construct copy in known orientation at a predetermined site 5′of the HPRT locus. HPRT expression renders the cell resistant to the toxic effects of hypoxanthine-aminopterin-thymidine media. This strategy avoids the necessity of introducing one or more selectable markers in the targeting construct that often have deleterious effects on subsequent levels and patterns of transgene expression. Moreover, the HPRT locus has proven to be exquisitely permissive for several promoter and enhancer combinations that drive transgene expression, including inducible Cre recombinase, in a wide variety of cell types like heart, muscle, kidney,etc.
Creative Animodel has constructed systematic data and experience in generating HPRT and Rosa26 knockin mouse models. Besides mouse models, we also provide rat/rabbit HPRT and Rosa26 knockin models. If you are interested in our laboratory, please feel free to contact us. Our research scientists will work closely with you to develop detailed study protocols to meet your goals.
The Workflow of HPRT and Rosa26 Knockin Mouse
1. HPRT/Rosa26 Knockin Mouse Strategy Design
* Our research scientists will work closely with you to develop detailed study protocols to meet your goals
* Targeted gene analysis and determination for the founder screening by using online prediction tools and bioinformatics software
* Targeting embryos or embryonic stem(ES) cells preparation
2. HPRT/Rosa26 Knockin Mouse Vector Construct
* Knockin mouse vectors to construct
* Constructed vector electroporates into targeted ES cells effectively
* PCR, Southern Blot and DNA sequencing to identify recombinant ES cells
3. Pronuclear Injection or ES cell targeting
1) Pronuclear injection
2) Es cell targeting
* Recombinant ES cells for blastocyst injections
* Blastocyst implanted into pseudopregnant surrogate mouse
4．HPRT/Rosa26 Knockin Mouse Screening
* Chimera mouse generation and screening
Our customer service representatives are available 24 hours a day, from Monday to Sunday.