STOCK Tg(Nkx2-5-tTA,tetO-EGFP/cre)1Smwu/J

Stock number: 029490
Product name: Nkx2.5 enh-GFP-Cre
Research areas: Cancer Research, Cardiovascular Research, Research Tools

Description

The doxycycline-regulated Nkx2.5 enhancer-eGFP/Cre transgenic mice have the cardiac-specific mouse NK2 homeobox 5 promoter/enhancer sequences directing dox-controllable expression of eGFP/Cre fusion protein to early heart progenitor cells (cardiomyoblasts) from embryonic day (E)7.5-E11.5. These Nkx2.5 enh-GFP-Cre transgenic mice allow Nkx2.5+ cardiomyoblast fluorescent labeling (cell isolation by FACS) and lineage tracing in a doxycycline-regulated fashion.
Of note, the donating investigator also has their Nkx2.5 enh-eGFP transgenic mice available as Stock No. 029489).

Detailed description

The Nkx2.5 enhancer-eGFP/Cre transgene (Nkx2.5 enh-GFP-Cre or Nkx2.5 enh-Cre) has doxycycline-regulatable expression of an eGFP/Cre fusion protein directed to the developing heart by the cardiac-specific mouse NK2 homeobox 5 promoter/enhancer sequences. Because the transgene contains both the tetracycline-controlled transactivator protein (tTA) as well as the tetracycline operator (tetO; also called tetracycline-responsive element [TRE] or tet-operator), expression of the downstream eGFP/Cre fusion protein can be blocked by the tetracycline analog doxycycline (dox). The donating investigator reports in the absence of dox, the fusion protein expression pattern (eGFP immunofluorescence/direct fluorescence, along with Cre recombinase activity) is specific to early heart progenitor cells from embryonic day (E)7.5-E11.5. Direct eGFP fluorescence is observable by whole mount fluorescence microscopy from E8.5 until E11.5, but only by flow cytometry at E7.5. Expression in neonatal heart is weak and difficult to detect. Immunohistochemical analysis reveals the presence of eGFP+ cells precisely in the myocardial layer with limited expression in the endocardial layers - identifying Nkx2.5 enh-GFP-Cre+ cells within the heart as cardiomyoblasts. Fully functional Cre recombinase activity follows the same pattern (as measured by expression of the Cre-inducible fluorescent reporter mT/mG [Stock Nos. 007576 or 007676]). The donating investigator reports dox-inducible cessation of eGFP/Cre expression is generally robust. eGFP/Cre expression may be prevented in pregnant females and all offspring by dox treatment starting at conception (1 mg/mL dox in drinking water).

To date (August 2016), it has not been determined if transgenic mice are homozygous or hemizygous. The donating investigator reports that transgenic mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. They report the eGFP/Cre expression has some variation between litters and even within a liter.

Comparing dox-regulated Nkx2.5 enh-GFP-Cre (Stock No. 029490) to Nkx2.5 enh-eGFP (Stock No. 029489): Overall, the donating investigator reports that the Nkx2.5 enh-GFP-Cre (before dox) expression pattern matches that of their Nkx2.5 enh-eGFP transgenic mice. However, the eGFP expression is much brighter in Nkx2.5 enh-eGFP compared to Nkx2.5 enh-GFP-Cre (before dox).

Development

The doxycycline-regulated Nkx2.5 enhancer-eGFP/Cre transgene (Nkx2.5 enh-GFP-Cre or Nkx2.5 enh-Cre) was created by Dr. Sean M. Wu (Stanford University School of Medicine). The transgene was designed with (from 5' to 3') with a mouse NK2 homeobox 5 locus (Nkx2-5) promoter/enhancer sequence (2.1 kbp enhancer fragment from the regulatory region ~9.5 kbp upstream of the translation start site and the 0.5 kbp base promoter sequence), a Tet-Off cassette (described below), an enhanced green fluorescent protein/Cre recombinase fusion gene sequence (eGFP/Cre) and a G-CSF polyA sequence. The Tet-Off cassette contains a tetracycline-regulated transactivator sequence (tTA), 2x polyA sequence and tetracycline-responsive element (TRE [tet_o] with minimal cytomegalovirus promoter [CMV_min]). The ~7.52 kbp transgene was injected into C57BL/6NCrl mice. Founder animals were bred to C57BL/6N mice for germline transmission, and a founder line was established. The Nkx2.5 enh-GFP-Cre colony was maintained on a mixed genetic background (including C57BL/6N, CD1 and 129Sv). In 2016, transgenic males with agouti coat color were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).
The donating investigator reports the transgene insertion site(s) is not known and there is no evidence of multiple copies.

Allele

Symbol: Tg(Nkx2-5-tTA,tetO-EGFP/cre)1Smwu
Name: transgene insertion 1, Sean Wu
MGI accession ID: MGI:5788432
Generation method: Transgenic
Attributes: Recombinase-expressing; Reporter; Inducible
Synonyms: Nkx2.5 enh-Cre
Marker symbol: Tg(Nkx2-5-tTA,tetO-EGFP/cre)1Smwu
Marker name: transgene insertion 1, Sean Wu
Chromosome: UN
Strain of origin: C57BL/6NCrl
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli; tetO,tet operator,; GFP/cre,Green Fluorescent Protein/Cre recombinase fusion protein,
Site of expression: In the absence of dox, the fusion protein expression pattern (eGFP immunofluorescence/direct fluorescence, along with Cre recombinase activity) is specific to early heart progenitor cells from embryonic day (E)7.5-E11.5.
Molecular note: The transgene was designed with (from 5' to 3') with a mouse NK2 homeobox 5 locus (Nkx2-5) promoter/enhancer sequence (2.1 kbp enhancer fragment from the regulatory region ~9.5 kbp upstream of the translation start site and the 0.5 kbp base promoter sequence), a Tet-Off cassette, an enhanced green fluorescent protein/Cre recombinase fusion gene sequence (eGFP/Cre) and a G-CSF polyA sequence. The Tet-Off cassette contains a tetracycline-regulated transactivator sequence (tTA), 2x polyA sequence and tetracycline-responsive element (TRE with minimal cytomegalovirus promoter CMVmin).