Mesp1BAC-mCherry transgenic mice were designed in the laboratory of Dr. Michael I. Kotlikoff (Cornell University) as part of Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).
The ~156 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP24-353L5 was obtained; containing the entire mesoderm posterior 1 locus (Mesp1) as well as ~23 kbp centromeric flanking sequences (including a portion of the Wdr93 locus) and ~131 kbp distal flanking sequences (including the complete Mesp2, Anpep and Ap3s2 loci).
Using homologous recombination/BAC recombineering, a construct containing the mCherry sequence, an SV40 polyadenylation signal, an SV40-puro-pA cassette and flanking vector sequences was all inserted into the ATG start site of the BAC Mesp1 gene (replacing the initiation codon of Mesp1 in exon 1). No other loci on the BAC were altered.
The resulting ~165 kbp modified BAC (Mesp1BAC-mCherry-pA-SV40-puro-pA) was purified and then microinjected into FVB/N embryos.
Founder animals were bred to C57BL/6J inbred mice for germline transmission. The donating investigator reports that Mesp1BAC-mCherry founder line 5 was backcrossed two generations to C57BL/6J wildtype mice, then bred together six generations as homozygotes, and then backcrossed four more generations to C57BL/6J wildtype mice. See SNP results below.
In 2015, mice were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).
In 2015, a 32 SNP (single nucleotide polymorphism) panel analysis was performed on the two male mice first received at The Jackson Laboratory Repository. This panel has 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains. Importantly, those latter 5 markers are the same for C57BL/6N and FVB. The SNP panel revealed 4 of 27 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for FVB allele-type markers); specifically 2 markers on chromosome 13 (~21 Mbp) in both mice, with each mouse also having one other C57BL/6;FVB mix marker (on chromosome 9 [~6.6 cM] or chromosome 10 [~87 Mbp]). In addition, 2 of the 5 markers that determine C57BL/6J from C57BL/6N (FVB) revealed both mice were segregating on chromosome 13 [~41.0 Mbp], with one mouse also segregating on chromosome 11 [~4 Mbp]. Collectively, these data suggest the mice sent to The Jackson Laboratory were not fully backcrossed onto C57BL/6, and are instead on a C57BL/6;FVB mixed genetic background.
