STOCK Tg(tetO-MAML1*/EGFP)2Akar/J

Stock number: 017918
Product name: TetOS-dnMAML-GFP
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

Tet^OS-dnMAML-GFP transgenic mice allow Tet-Off/Tet-On expression of a dominant negative form of MAML, and may be may be useful for temporally-controlled, tissue-specific blockade of Notch signaling.

Detailed description

The Tet^OS-dnMAML-GFP transgene has the Tet response element (TRE) upstream of a cDNA sequence encoding a dominant-negative mutant variant of human Mastermind-like 1 (dnMAML) fused in-frame at its C-terminus to an enhanced green fluorescent protein (EGFP). The dnMAML-GFP fusion protein [also called DNMAML1-GFP or MAML1(13-74)-GFP] interrupts transactivation/nuclear activity of all four Notch receptors by binding the Notch-CSL [CBF1/Su(H)/Lag2 protein] transcriptional complex, but does not bind the coactivators required for transcription of target genes.

Mice homozygous for the Tet^OS-dnMAML-GFP transgene are viable and fertile with no reported phenotypic abnormalities. When bred with other mice expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), dnMAML-GFP expression in the resulting double mutant offspring can be regulated with tetracycline or its analog doxycycline (dox). When tTA or rtTA(+dox) is present, dnMAML expression blocks the transactivation/nuclear activity of all four Notch genes by binding to the Notch-CSL [CBF1/Su(H)/Lag2 protein] transcriptional complex (preventing Notch-CSL from binding its endogenous coactivator, MAML), and results in a Notch quadruple knockout in the tTA-expressing cells. GFP expression be observed by direct/native fluorescence and antibody staining/immunohistochemistry (the donating investigator did not attempt mRNA (in situ) hybridization). As designed, Tet^OS-dnMAML-GFP transgenic mice have no reported levels of dnMAML-GFP expression in the absence of tTA.

These Tet^OS-dnMAML-GFP transgenic mice allow Tet-Off/Tet-On expression of a dominant negative form of MAML, and may be may be useful for temporally-controlled, tissue-specific blockade of Notch signaling. For example, when bred to a strain expressing tTA in blood vessel endothelial cells (VE^tTA: Stock No. 013585), the resulting double mutant mice are useful for identifying Notch target genes in endothelial-to-mesenchymal transdifferentiation in cardiac development.

Development

The Tet^OS-dnMAML-GFP transgene was designed with a TetOS promoter followed by a cDNA sequence encoding the dnMAML-GFP fusion protein and an SV40 polyA sequence. The TetOS promoter (Tet response element or TRE) contains seven copies of the 42-bp tet operator sequence (tetO) just upstream of a minimal CMV promoter. The dnMAML-GFP cDNA sequence encodes an N-terminal, 62 amino acid (amino acids 13-74), dominant-negative mutant variant of human Mastermind-like 1 (dnMAML) fused in-frame at its C-terminus to an enhanced green fluorescent protein (EGFP). The ~3.0 kbp Tet^OS-dnMAML-GFP transgene was injected into the pronucleus of ICR oocytes fertilized by ICR males. Founder mice were bred with ICR mice to establish founder line 2. The colony was maintained by breeding Tet^OS-dnMAML-GFP transgenic mice with ICR mice for several generations, and then by breeding Tet^OS-dnMAML-GFP transgenic mice with CD-1 outbred mice for several generations prior to sending males to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664).

Allele

Symbol: Tg(tetO-MAML1*/EGFP)2Akar
Name: transgene insertion 2, Aly Karsan
MGI accession ID: MGI:5496022
Generation method: Transgenic
Attributes: Reporter; Inducible; Dominant negative; Inserted expressed sequence
Marker symbol: Tg(tetO-MAML1*/EGFP)2Akar
Marker name: transgene insertion 2, Aly Karsan
Chromosome: UN
Strain of origin: ICR
Expressed genes: MAML1,mastermind like transcriptional coactivator 1,human; GFP,Green Fluorescent Protein,
Site of expression: These transgenic mice allow Tet-Off/Tet-On expression of a dominant negative form of MAML, and may be may be useful for temporally-controlled, tissue-specific blockade of Notch signaling. For example, when bred to a strain expressing tTA in blood vessel endothelial cells the resulting double mutant mice are useful for identifying Notch target genes in endothelial-to-mesenchymal transdifferentiation in cardiac development.
Molecular note: The Tet-dnMAML-GFP transgene was designed with a TetOS promoter followed by a cDNA sequence encoding the dnMAML-GFP fusion protein and an SV40 polyA sequence. The TetOS promoter (Tet response element or TRE) contains seven copies of the 42-bp tet operator sequence (tetO) just upstream of a minimal CMV promoter. The dnMAML-GFP cDNA sequence encodes an N-terminal, 62 amino acid (amino acids 13-74), a dominant-negative mutant variant of human Mastermind-like 1 (dnMAML) fused in-frame at its C-terminus to an enhanced green fluorescent protein (EGFP).