FVB(C)-Tg(tetO-Npc1/YFP)1Mps/J

Stock number: 021065
Research areas: Neurobiology Research, Research Tools

Description

YFP-labeled NPC1 (Niemann Pick type C1) protein expression is conditionally directed via a tetracycline-inducible minimal cytomegalovirus (tetO-CMV_min) promoter in this transgenic strain. When bred with mice expressing reverse tetracycline-controlled transactivator protein (rtTA) or tetracycline-controlled transactivator protein (tTA), NPC1-YFP expression can be regulated with the tetracycline analog doxycycline (dox) in the resulting double mutant offspring.

Detailed description

Loss of NPC1 (Niemann Pick type C1) protein function is a known cause of Niemann-Pick type C disease, a fatal lysosomal storage disorder associated with progressive, widespread neurological deterioration. Symptoms include, but are not limited to, dementia, ataxia, dysarthria, dystonia, and epilepsy.

In this transgenic strain, expression of YFP-labeled and FLAG-tagged NPC1 is conditionally directed via a tetracycline-inducible minimal cytomegalovirus (tetO-CMV_min) promoter. When bred with mice expressing reverse tetracycline-controlled transactivator protein (rtTA) or tetracycline-controlled transactivator protein (tTA), NPC1-YFP expression can be regulated with the tetracycline analog doxycycline (dox) in the resulting double mutant offspring. Expression of the transgene is undetectable in heterozygotes in the absence of a tTA/rtTA driver.

Founder lines were test-crossed with Pcp2-tTA (Purkinje cell protein 2 (L7)) driver animals (see Stock No. 005625) to select a transgenic line that best exhibited induction of NPC1-YFP in Purkinje neuron layer of the mouse cerebellum.

When tetO/tTA (or tetO/rtTA) double mutants are further combined with an Npc1 knockout (see Stock No. 021755), cell type-specific rescue of NPC1 loss can be tested. NPC1-YFP produced in cerebellar Purkinje neurons of Npc1 knockout mice prevents neuron degeneration, slows reactive glial activity, and ameliorates disease. These mice show reduced ataxia, increased weight, and prolonged life, but the transgene does not prevent the eventual decline and premature death of knockout mice. Cell types responsible for disease pathogenesis can be traced through the fluorescent signal.

Robust expression of NPC1-YFP in skin and visceral tissue is observed in offspring generated through crosses with B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} (see Stock No. 006965) and treated with dox.

Development

A transgenic vector containing Npc1-6xHis-YFP-2xFLAG driven by a tet operon-cytomegalovirus minimal promoter (tetO-CMV_min) was introduced to FVB/N embryos. Founder animals were test-crossed with FVB-Tg(Pcp2-tTA)3Horr/J driver animals (see Stock No. 005625) to select a transgenic line that best exhibited induction of NPC1-YFP in the Purkinje neuron (PN) layer of the mouse cerebellum. Pcp2-tTA was subsequently bred away from this strain.

Transgenic animals were crossed with FVB.C-Npc1^m1N (Stock No. 021755) heterozygotes (background then N5-6 FVB). The compound mutant mice were backcrossed crossed 2-3 times to FVB/NJ. Animals lacking the Npc1^m1N knockout mutation were selected from the donating lab's (Tg Homozygous, m1N Heterozygous) colony to generate this single-mutation transgenic line.

The transgenic insertion site was mapped by 3' end genome walking. The 3' end is directly before a PstI restriction enzyme site at Chr4:36,629,314. A HindIII site is located nearby at position Chr4:36,631,149. The 5' integration end is not known, nor is the transgenic copy number.

Allele

Symbol: Tg(tetO-Npc1/YFP)1Mps
Name: transgene insertion 1, Matthew Scott
MGI accession ID: MGI:5468713
Generation method: Transgenic
Attributes: Reporter; Inducible; Inserted expressed sequence
Marker symbol: Tg(tetO-Npc1/YFP)1Mps
Marker name: transgene insertion 1, Matthew Scott
Chromosome: 4
Strain of origin: FVB/N
Expressed genes: Npc1,NPC intracellular cholesterol transporter 1,mouse, laboratory; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: In this transgenic strain, expression of YFP-labeled and FLAG-tagged NPC1 is conditionally directed via a tetracycline-inducible minimal cytomegalovirus (tetO-CMV_min) promoter. When bred with mice expressing reverse tetracycline-controlled transactivator protein (rtTA) or tetracycline-controlled transactivator protein (tTA), NPC1-YFP expression can be regulated with the tetracycline analog doxycycline (dox) in the resulting double mutant offspring. For example, bred with mice carrying the Eno2-tTA trans-gene drives reporter expression in neurons throughout the central nervous system.
Molecular note: The transgenic vector contains a Npc1-6xHis-YFP-2xFLAG driven by a tet operon-cytomegalovirus minimal promoter (tetO-CMVmin). Line 1 was generated.