These knock-in mice express Lin28a under the regulatory control of a Col1a1-driven tetO promoter. These mice are larger than non-transgenic animals, even in the absence of tTA/rtTA, show delayed estrus, and a resistance to age/diet-induced obesity.
George Q. Daley, Children's Hospital Boston
LIN28A (lin-28 homolog A (C. elegans)) is an RNA-binding protein that blocks biogenesis of Mirlet7 (let-7) microRNAs. Let-7 targets are known regulators of mammalian body size and metabolism.
These knock-in mice express Lin28a under the regulatory control of a Col1a1 (collagen, type I, alpha 1)-driven tetO promoter. These mice are larger than non-transgenic animals, show wider faces, larger ears, and coarser hair, even in the absence of tTA/rtTA/doxycyline induction. RT-PCR demonstrates that there is some ectopic expression of the transgene in both neonates and adults. In adults, there is a sevenfold increase in the level of Lin28a mRNA in skeletal muscle, a fivefold increase in the ovary, and fourfold increase in the hypothalamus.
Fasting and fed glucose levels are lower in transgenic mice. Both males and females clear glucose more efficiently. Transgenic mice also have increased insulin sensitivity and are resistant to age-induced and diet-induced obesity, as well as hepatosteatosis. Mice show markedly improved glucose tolerance and insulin sensitivity under high fat diet conditions. Transgenic mice achieve first estrus at day 31.8, as compared to day 27.3 in wildtype mice. Transgenic animals show an approximated 3 day delay to the date of the first litter. Overall fertility in mutant and wildtype mice is about the same over the first 3 months of life.
Lin28a expression can be induced with doxycycline in progeny derived from a cross with a Gt(ROSA)26Sor M2-rtTA strain (see Stock No. 006965). This leads to rapid death associated with marked gut pathology. Uninduced mice have thickened skin, larger bones and proportionately enlarged visceral organs.
Uninduced Gt(ROSA)26Sor M2-rtTA/tetO-Lin28a compound mutant mice are said to have the same phenotype as animals carrying only the tetO-Lin28a mutation.
A targeting vector containing a splice acceptor, 2 polyadenylation sequences, a tetracycline operator, flag-tagged mouse Lin28a cDNA, and an SV40 polyadenylation signal within 3'UTR of the mouse Col1a1 gene was introduced to (C57BL/6 x 129S4/SvJae)F1-derived v6.5 embryonic stem (ES) cells containing M2-rtTA (optimized rtTA) targeted to the Gt(ROSA)26Sor locus promoter (see Stock No. 006965 for mutation details). Resultant chimeric males were crossed with outbred CD1 females. The Gt(ROSA)26Sor M2-rtTA mutation was bred away from this strain. The line was backcrossed to C57BL/6 for 8 generations by the donating lab (see SNP note below).
A 32 SNP (single nucleotide polymorphism) panel analysis, with 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, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
|Allele Name||targeted mutation 1, George Daley|
|Allele Type||Targeted (Inducible, Inserted expressed sequence, RMCE-ready)|
|Allele Synonym(s)||Lin28a Tg|
|Gene Symbol and Name||Col1a1, collagen, type I, alpha 1|
|Promoter||tetO, tet operator,|
|Strain of Origin||(C57BL/6 x 129S4/SvJae)F1|
|General Note||The allele was made in KH2 cells that carry Gt(ROSA)26Sortm1(rtTA*M2)Jae and Col1a1tm13(neo/hygro*))Jae. This allele was generated with Col1a1tm13(neo/hygro*)Jae.|
|Molecular Note||RMCE on KH2 cells inserted a cassette that contains a splice acceptor, 2 polyadenylation sequence, tetracycline operator, flag-tagged mouse cDNA, and an SV40 polyadenylation signal into the 3'UTR. Leaky ectopic expression in the absence of doxycycline induction was confirmed by quantitative RT-PCR and immunohistochemistry.|
Homozygous and heterozygous tetO-Lin28a are viable and fertile.
When using the B6.Cg-Col1a1tm1(tetO-Lin28a)Gqda/J mouse strain in a publication, please cite the originating article(s) and include JAX stock #023910 in your Materials and Methods section.