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Mouse chromosome 3 sequence was retrieved from Ensembl database build 30. Mouse C57BL/6 BAC clone RP23-4L11 was used to generate the homologous arms and the conditional KO region for the gene-targeting vector and the southern probes for screening-targeted events. PCR or REDcloning/gap-repair methods were used to clone the appropriate sequences. The 5’homologous arm (5.6 kb) and conditional knockout region (3.8 kb) were generated by RED cloning/gap repair. The 3= homologous arm (3.9 kb) was generated by PCR reaction using proofreading LA Taq DNA polymerase. These fragments were cloned into the pCRXL vector and confirmed by restriction digest and end sequencing. The final vector also contains loxP sequences flanking the conditional knockout region, loxP sequences flanking the Neo expression cassette (for positive selection of the ES cells), and a DTA expression cassette (for negative expression of the ES cells). The final vector was confirmed by both restriction digestion and end sequencing analysis. Notl was used to linearize the final vector before electroporation. Figure 1A ofPMID:20719974 shows the key features of the final vector. 5’ and 3’ external probes were generated by PCR reaction and tested on genomic Southern analysis for ES screening. They were cloned in pCR2.1 backbone and confirmed by sequencing. Subsequently, ES cells were transfected with the gene-targeting construct and homologous recombinant clones were identified. The ES cells were derived from a C57BL/6 Tac inbred mouse strain. After successful homologous recombination, the donor electroporated a supercoiled Neo expression plasmid into the correctly targeted ES clones. Under no G418 selection, ES clones were picked. G418 sensitivity was tested on duplicates of these clones. PCR was then performed to confirm the Neo deletion on the G418-sensitive clones. Positive ES clones were injected into blastocysts of FVB/NTac recipient mice to create chimeras. High-percentage chimeras were bred with C57BL/6 Tac to generate heterozygous floxed Rhbg mice, which were then used to generate homozygous floxed Rhbg mice.
Bishop JM, Verlander JW, Lee HW, Nelson RD, Weiner AJ, Handlogten ME, WeinerID. Role of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammoniaexcretion. Am J Physiol Renal Physiol. 2010 Nov;299(5):F1065-77. doi:10.1152/ajprenal.00277.2010. Epub 2010 Aug 18. (Medline PMID: 20719974)
Colony Surveillance Program and Current Health Reports
Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.
Cryopreserved material may be available upon request, please inquire to mmrrc@missouri.edu for more information.
The donor or their institution limits the distribution to non-profit institutions only.
Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.
Additional charges may apply for any special requests. Shipping costs are in addition to the basic distribution/resuscitation fees. Information on shipping costs and any additional charges will be provided by the supplying MMRRC facility.
1 The distribution fee covers the expense of rederiving mice from a live mouse; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.
2 An aliquot contains a sufficient number of embryos (in one or more vials or straws and based on the transfer success rate of the MMRRC facility) to transfer into one to three recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.
3 An aliquot is one straw or vial with sufficient sperm to recover at least one litter of mice, as per provided protocols, when performed at the MMRRC facility. The MMRRC makes no guarantee concerning the success of these procedures when performed outside the MMRRC facilities.
4 The distribution fee covers the expense of resuscitating mice from the cryo-archive; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.