Mice heterozygous for the NSML knock-in allele (NSML/+ or Ptpn11Y279C/+; formerly called LS/+) have the Ptpn11*Y279C mutation in exon 7 that is one of the two most common mutations associated with Noonan Syndrome with Multiple Lentigines (NSML; formerly called Leopard syndrome [LS]). These mice recapitulate several characteristics of the human disorder, including hypertrophic cardiomyopathy, short stature, craniofacial dysmorphia, abnormal genitalia and substantial macrophage accumulation in the organ of Corti.
Maria I Kontaridis, Beth Israel Deaconess Medical Center
Noonan Syndrome with Multiple Lentigines (NSML; formerly called Leopard syndrome [LS]) is an autosomal dominant "RASopathy" characterized by multiple brown skin spots (lentigines), congenital heart disease, short stature, genital abnormalities, hearing loss and distinctive facial features. In humans, the PTPN11*Y279C mutation is one of the two most common NSML mutations.
The ubiquitously expressing (non-inducible) NSML knock-in allele (Ptpn11Y279C) encodes the Ptpn11*Y279C mutant protein. Mice heterozygous for this allele (Ptpn11Y279C/+ or NSML/+; formerly LS/+) recapitulate several characteristics of the human disorder. Morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy is evident by 12 weeks of age.
NSML/+ mice may also exhibit any of the following at variable penetrance: craniofacial dysmorphia, skeletal/chest abnormalities, shorter stature compared to wildtype mice. Heterozygous mice exhibit premature lethality (~25% die between 8-12 months of age), and surviving NSML/+ mice of both sexes are viable and fertile to at least 12 months of age.
In addition, heterozygotes have abnormal genitalia and substantial macrophage accumulation in the organ of Corti (inner ear). Importantly, treatment with the mTOR-inhibitor rapamycin effectively reverses the NSML disease phenotype.
Specifically, NSML/+ mice have hypertelorism appearance (abnormal faces, with distinctly smaller, more slanted eyes and a planar nasal bridge). Heart and/or cardiomyocyte lysates from NSML/+ mice show enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. NSML/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The donating investigator reports they keep breeding units between 4-8 months of age, and abnormal genitalia can sometimes delay breeding by heterozygous males.
Expression of this Ptpn11Y279C knock-in allele (both mRNA and protein) is at levels comparable to wildtype Ptpn11. Peripheral blood, bone marrow, spleen, and thymus from NSML/+ mice show no apparent abnormalities in any hematological parameter.
The donating investigator reports that homozygous mice (Ptpn11Y279C/Y279C or NSML/NSML; formerly LS/LS) exhibit a more severe phenotype and ~50% of homozygotes mice die between 8-12 months of age. Homozygous mice are born at significantly reduced frequency, and homozygous females do not lactate.
The Ptpn11Y279C knock-in allele has the Ptpn11*Y279C mutation in exon 7 that is one of the two most common mutations associated with Noonan Syndrome with Multiple Lentigines (NSML; formerly called Leopard syndrome [LS]). This knock-in allele was created by Dr. Maria I. Kontaridis (Beth Israel Deaconess Medical Center). First, the inducible NSML ("iLS") targeting vector was designed to modify the protein tyrosine phosphatase, non-receptor type 11 gene (Ptpn11 or Shp2) on chromosome 5 with (from 5prime to 3prime) a loxP site upstream of endogenous exon 7, a loxP511 site, an inverted Y279C mutant exon 7 (encoding an amino acid substitution of cysteine for tyrosine at position 279), an inverted loxP site, an inverted loxP511 site, a frt-flanked neo cassette. This also inserted two HpyCH4 restriction sites (intended to be useful in screening/genotyping): one in the mutant exon 7 Y279C site and the other at the 5prime end of the inserted targeted mutant sequence 35 bp downstream of the 5prime loxP511 site. The construct was electroporated into 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells.
Chimeric mice were bred to C57BL/6J for germline transmission. The resulting iLS/+ mice (129S6/SvEv x C57BL6/J) were crossed to Flp recombinase-expressing mice on a C57BL/6J genetic background (Stock No. 005703) to excise the frt-flanked neo cassette. Next, the mice were bred to Cre recombinase-expressing mice on a C57BL/6J genetic background (Stock No. 003724) to remove the floxed endogenous exon 7 and place the Y279C mutant exon 7 into transcriptional orientation; resulting in the ubiquitously expressing (non-inducible) NSML allele ("LS"). The FLP and Cre transgenes were selectively removed in future breedings.
Heterozygous mice (Ptpn11Y279C/+ or NSML/+; formerly LS/+) were bred with wildtype C57BL/6J mice for at least nine generations prior to sending black male mice in 2014 to Frederick National Laboratory for Cancer Research (Leidos Biomedical Research, Inc.). There, the NSML/+ mice were bred with C57BL/6J females at least one generation. In 2015, black male mice were then sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To generate the living NSML colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). The NSML colony was then maintained by breeding heterozygous mice to wildtype mice from the colony or with C57BL/6J inbred mice.
|Allele Name||targeted mutation 4.2, Benjamin G Neel|
|Allele Type||Targeted (Humanized sequence)|
|Allele Synonym(s)||Ptpn11LS; Ptpn11Y279C|
|Gene Symbol and Name||Ptpn11, protein tyrosine phosphatase, non-receptor type 11|
|Strain of Origin||129S6/SvEvTac|
|Molecular Note||A loxP site was inserted upstream of exon 7. An inverted modified exon 7 with a nucleotide substitutions that result in the amino acid substitution of cysteine for tyrosine at position 279 (Y279C) with a 5' loxP site was flanked by loxP511 sites and inserted downstream of exon 7 along with an FRT flanked neo cassette. Flp-mediated recombination removed the neo cassette, and cre-mediated recombination removed the endogenous exon 7 and reverted the orientation of the modified exon 7. The amino acid substitution is one of the two most common mutations in Leopard syndrome.|
When maintaining our live colony, heterozygous females are bred to wildtype males from the colony or with C57BL/6J inbred males (Stock No. 000664).
Heterozygous mice (both female and male) are viable and fertile to at least 12 months of age. ~25% of heterozygotes die between 8-12 months of age. The donating investigator reports they keep breeding units between 4-8 months of age, and abnormal genitalia can sometimes delay breeding by heterozygous males. Homozygous mice exhibit a more severe phenotype; ~50% die between 8-12 months of age and females do not lactate. Homozygotes are born at significantly reduced frequency.
When using the Noonan Syndrome with Multiple Lentigines knock-in (NSML/+ or Ptpn11Y279C/+; formerly called Leopard syndrome knock-in [LS/+]) mouse strain in a publication, please cite the originating article(s) and include JAX stock #026759 in your Materials and Methods section.
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