Mice homozygous for this Tgfb1flox-ex 6 conditional allele are viable and fertile, with loxP sites flanking exon 6 of the Tgfb1 (transforming growth factor, beta 1) gene. When bred to mice that express Cre recombinase, the resulting offspring will have the floxed region deleted in the cre-expressing tissues while leaving the precursor pro-region and neighboring B9d2 (LOC232987) gene intact.
In response to some users reporting less cre-induced recombination than expected, the mutation in this line was sequenced. The 3' loxP site contains an anomaly, which could decrease the efficiency of the cre recombinase used. The loxP sites are in an orientation that results in an inversion, not a deletion.
A targeting vector was designed to place a loxP-flanked PMC1-Neo cassette just upstream of exon 6, and a third loxP site just downstream of exon 6 of the targeted gene. This construct was electroporated into 129S6/SvEvTac-derived KG-1 embryonic stem (ES) cells. Correctly targeted ES cells were then transiently transfected with a Cre recombinase-expressing plasmid. The resulting ES cells with the Tgfb1flox-ex 6 genotype (neo selection cassette removed; leaving a single loxP site upstream of exon 6 and a single loxP site downstream of exon 6) were injected into recipient blastocysts. Chimeric males were bred with Black-Swiss females to generate the Tgfb1flox-ex 6 colony. Homozygous mice were bred together for approximately five generations and then bred with Tgfb1- mice (on a mixed CF-1, 129 and BALB/c genetic background; see Stock No. 002098). The Tgfb1flox-ex 6/- mice were bred together for approximately ten generations. Offspring homozygous for the Tgfb1flox-ex 6 allele were sent to The Jackson Laboratory. Upon arrival, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the Tgfb1flox-ex 6 colony.
In September 2011, the living Tgfb1flox-ex 6 mouse colony at The Jackson Laboratory Repository was found to be homozygous for the Tgfb1flox-ex 6 allele and to also be segregating for the Cre recombinase gene. The Cre recombinase expression was unlikely to be in germline (as the colony was homozygous for the Tgfb1flox-ex 6 allele using an assay that would not produce a PCR product for a deletion allele). Whether or not the source of Cre recombinase was from previously mating Tgfb1flox-ex 6 mice with LckCre mice is not known. Mice double confirmed to be Cre-negative were selected and bred to C57BL/6J inbred mice to repopulate the colony. By May 2012, the living Tgfb1flox-ex 6 mouse colony at The Jackson Laboratory Repository was homozygous for the Tgfb1flox-ex 6 mutation and without Cre recombinase.
Further investigation has revealed that the 3' loxP site contains an anomaly, which could decrease the efficiency of the cre recombinase used. The loxP sites are in an orientation that results in an inversion, not a deletion.
|Allele Name||targeted mutation 2.1, Thomas Doetschman|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Allele Synonym(s)||Tgfb1flox ex6|
|Gene Symbol and Name||Tgfb1, transforming growth factor, beta 1|
|Strain of Origin||129S6/SvEvTac|
|Molecular Note||A floxed neomycin cassette was placed upstream of exon 6 with an additional loxP site placed downstream of exon 6. The neomycin cassette was subsequently removed by transient expression of cre recombinase leaving exon 6 floxed. Germline transmission of the allele was confirmed by genomic PCR.|
|Mutations Made By|| |
Thomas Doetschman, University of Arizona