• Gene: murine Sell (MGI:98279)
• mRNA, variant 1: NM_011346.2
• DNA level: c.526_528delAACinsGGA
• protein level: p.Asp176Gly (N176G)

CJ7 derived from 129S1/Sv-Oca2⁺ Tyr⁺ Kitl⁺

An Entrez Gene search of GenBank revealed an 18-kb intact murine L-selectin genomic sequence. Exon 4 of the murine L-selectin gene encodes the Asn138 that is identical to its human counterpart. Genomic sequences around exon 4 were amplified by high-fidelity PCR using genomic DNA isolated from CJ7 murine embryonic stem cells. These sequences were used to prepare a knock-in targeting vector; site directed mutagenesis was used to substitute three nucleotides in the codon for Asn138 so that it now encoded Gly138. We designated the wild-type allele as “N” and the knock-in allele as “G” to indicate the amino acid encoded at position 138. Since it is unknown whether the mutant L-selectin would create major abnormalities during development or during early postnatal life, we also introduced a loxP-flanked neostop cassette fragment into intron 2. The neo portion of the cassette allows drug selection of transfected ES cells with G418, whereas the stop portion prevents downstream transcription. Finally, a thymidine kinase (tk) gene was inserted adjacent to the 3’-flanking homologous sequence of the targeting vector to enrich for targeted clones by negative selection with ganciclovir. The targeting vector was assembled in pBluescript, and the fidelity of the targeting construct was verified by DNA sequencing. The linearized targeting vector was electroporated into CJ7 murine embryonic stem cells. After G418 (300 µg/ml) and ganciclovir (2 µM) selection, 10/224 surviving clones with correct homologous recombination were identified by PCR and confirmed by Southern blotting; of these, 5/10 contained the 138G mutation. Heterozygous mutant ES cells from one clone confirmed to have a normal karyotype were microinjected into C57BL/6J blastocysts to generate chimeric mice. Germline transmission was achieved from two chimeric males with high percentages of coat color. The presence of the 138G mutation was established by PCR amplification of a 323 bp fragment from mouse tail genomic DNA with primers. L-selectinN138G heterozygous mice were mated with Cre deleter mice E2a-Cre. Cre recombinase, under the control of the adenovirus EIIa promoter, expressed at the one-cell zygote stage, deletes the neostop cassette early during development. The first generation from this breeding was screened for loss of neostop cassette by PCR. PCR assay was designed to detect the 203 bp band from wild-type allele and 340 bp band from neostop-excised allele (called Δneostop), but could not amplify the mutant allele with neostop, which has not been excised by cre. The efficient excision was examined by PCR using primers to detect the neostop remaining at the targeted locus after cre. Heterozygous progeny that displayed complete neostop excision in the tail were selected for breeding back to C57BL/6J mice for more than 10 generations. N138G mutation and complete neostop excision among progeny were confirmed by PCR assay mentioned above and by DNA sequencing.

Wild-type littermates

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact mmrrc@missouri.edu. Older strains may not have this information.

McEver [M.D.], Rodger