Models

Vector Design and Construction

REM can design and construct gene targeting vector for use in ES cell experiments. Services offered include the isolation of gene fragments from murine genomic libraries, the construction of vectors to maximize targeting efficiency in ES cells, and the identification of DNA probes to confirm proper gene targeting in the ES cells and in mice. Alternatively, those clients with experience in vector design and construction may prefer to build the targeting vector and save the costs associated with these steps. In addition, REM (or the client) can design transgenes for non-homologous recombination into ES cells, permitting the development of transgenic mice by subsequent blastocyst injection of the ES cell clones.

Gene Targeting- Knockout mice

Classic knockout mice can be generated by replacement of one or more exons in a gene with a positive drug selection marker such as neomycin. Typically, a key exon is targeted for replacement in such a way as to leave the remainder of the coding sequences out of translational frame with the upstream coding sequences. This strategy reduces the likelihood of generating a hypomorphic mutation in the mice. The drug-resistant ES cell clones will be arrayed and screened for proper targeting. Expanded clones will be analyzed by Southern analysis to confirm correct targeting of the desired gene prior to their use in blastocyst injection experiments. These quality control steps help ensure that a true-knockout allele will be generated in the mice.

Gene Targeting- Knockin mice

Mice bearing subtle mutations in genes can be generated using knockin ES cell gene targeting strategies. The use of knockin vectors permits an investigator to place heterologous mammalian cDNA sequences or bacterial marker genes under transcriptional control of an endogenous promoter, to alter specific amino acid residues encoded by the target gene (e.g. "humanizing" a mouse gene), or to alter secondary protein modification targets (phosphorylation or acetylation sites) in the gene product. REM has previously generated knockin mice for each of these examples.

Gene Targeting- Conditional mice

Often the preferred targeting, the generation of conditional alleles in mice avoids the potential generation of an early embryonic lethal phenotype that hinders analysis of gene function in latter stages of development or in adult mouse. In the typical conditional allele, key exons in the gene of interest are surrounded by loxP sites through gene targeting experiments in ES cells. Subsequent exposure of the targeted allele to Cre recombinase will delete the sequences flanked by the loxP sites, thereby deleting the key exon(s) and generating a loss-of function allele. Expression of Cre in select tissue is achieved by breeding the conditional mice to transgenic mice that express Cre in a defined temporal or spatial manner. REM has extensive experience in designing conditional alleles in mice and is a licensed user of Cre-lox technology.

Generation of Chimeric Mice

REM utilizes highly efficient, 129SV-strain ES cells in gene targeting experiments to facilitate successful generation of high degree chimeric mice. Blastocysts microinjection experiments are performed using multiple clones of correctly targeted ES cells whenever possible. The chimeric mice are generated in a specific pathogen free facility. All mouse work is performed under SPF conditions, and monthly screenings are performed to ensure that the mice remain pathogen free. The chimeric mice can be bred by REM or can be shipped to the client for subsequent breeding, saving the client the costs associated with this step.

Breeding to achieve germline transmission

High degree chimeric mice are bred with wildtype mice (typically C57Bl/6 strain) to generate the F1 generation mice bearing the modified allele. Following weaning of the F1 litter, heterozygous mice are identified and shipped to the client. REM will retain some of the mice until the client has safely received the shipment. Other services, such as cryopreservation, are available upon request. Please note that REM does not maintain or further analyze your mice (unless otherwise specifically arranged) and does not offer your mice to other, third parties. In addition, REM is a contract service lab and does not retain any reach-through rights on your models.

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