B6;FVB-Tg(Mesp1-mCherry)5Mik/J

Stock number: 025618
Product name: CHROMus line Mesp1^BAC-mCherry
Research areas: Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

Mesp1^BAC-mCherry transgenic mice exhibit high mCherry fluorescence in mesodermal cell lineages during early gastrulation.

Detailed description

Mesp1^BAC-mCherry transgenic mice express the red fluorescent protein mCherry under control of the Mesp1 locus promoter/enhancer regions within the BAC transgene.

Mesp1^BAC-mCherry transgenic mice from founder line 5 exhibit mCherry expression (red fluorescence) in mesodermal cell lineages during early gastrulation, including cardiac mesoderm. In total, transgene expression is consistent with endogenous Mesp1.

Hemizygous and homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator reports that homozygotes have brighter fluorescence than hemizygotes.

This mouse model is available by way of a collaborative effort between Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus) and The Jackson Laboratory.

Development

Mesp1^BAC-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 (Mesp1^BAC-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 Mesp1^BAC-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.

Allele

Symbol: Tg(Mesp1-mCherry)5Mik
Name: transgene insertion 5, Michael I Kotlikoff
MGI accession ID: MGI:5693374
Generation method: Transgenic
Attributes: Reporter
Synonyms: Mesp1^BAC-mCherry
Marker symbol: Tg(Mesp1-mCherry)5Mik
Marker name: transgene insertion 5, Michael I Kotlikoff
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: Mesp2,mesoderm posterior 2,mouse, laboratory; Mesp1,mesoderm posterior 1,mouse, laboratory; Anpep,alanyl (membrane) aminopeptidase,mouse, laboratory; RFP,Red Fluorescent Protein,coral; Ap3s2,adaptor-related protein complex 3, sigma 2 subunit,mouse, laboratory
Site of expression: mCherry is expressed in mesodermal cell lineages during early gastrulation.
Molecular note: The ~165 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP24-353L5 contains 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. mCherry expression is observed in mesodermal cell lineages during early gastrulation, including cardiac mesoderm and is consistent with endogenous Mesp1.