129S;B6-Gt(ROSA)26Sor^{tm1.1(CAG-HL9-MG*V70A,-mBFP,-SNCA,-mTFP1,-SNCA*A30P,-mKO2,-SNCA*A53T)Rali}/J

Stock number: 037603
Product name: SNCAbow
Research areas: Neurobiology Research, Research Tools

Description

These α‐synuclein-Crainbow (SNCAbow) mice have a CAG promoter followed by three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP), and the SNCAbow construct, all targeted into the Gt(ROSA)26Sor locus. SNCAbow contains four fluorescent proteins, three of which are associated with three human α-synuclein protein sequences (SNCA^WT, SNCA^A30P, or SNCA^A53T), and functions as a Cre recombinase-inducible reporter of either a chemically inducible near‐infrared fluorogen‐activating peptide (FAPMars1) or one of the three nuclear localized fluorescent proteins. This strain may be useful for studying the role of α‐synuclein expression Parkinson's and related neurodegenerative diseases.

Detailed description

Expressed throughout the brain in neurons and enriched at presynapses, α-synuclein participates in synaptic vesicle function and represents a major component of the Lewy pathology found in Parkinson’s and related neurodegenerative diseases.

These SNCA mice have a CAG promoter followed by three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP), and the α‐synuclein-Crainbow construct, all targeted into the Gt(ROSA)26Sor locus (SNCAbow). Specifically, the SNCAbow construct contains three pairs of orthogonal lox sites that separate four cassettes, three of which are associated with a corresponding α-synuclein gene sequence. The first cassette expresses a chemically inducible near‐infrared fluorogen‐activating peptide (FAPMars1). The next three cassettes encode a unique nuclear localized fluorescent protein (TagBFP:blue, mTFP1:turquiose, or mKO2:orange) and a corresponding human α-synuclein protein sequence (SNCA^WT, SNCA^A30P, or SNCA^A53T, respectively) each separated by a P2A peptide to mediate ribosomal skipping. Each α-synuclein sequence contains a C-terminal epitope tag: SNCA^WT tagged with V5, SNCA^A30P tagged with 3XHA, or SNCA^A53T tagged with Myc. To ensure that only one fluorescent protein is expressed at any one time, an SV40 polyadenylation signal was inserted downstream of each open reading frame. In the absence of Cre recombinase activity, only the near-infrared fluorogen-activating protein (FAP) is expressed. In the presence of Cre recombinase, mice express either nuclear localized SNCA^WT:TagBFP, SNCA^A30P:mTFP1, or SNCA^A53T:mKO2 in cre-expressing tissues of the offspring.

Of note, when bred to Vil‐CreERT2 mice (Stock No. 020282), resulting SNCAbow;Vil‐CreERT2 mice allow for timed inducible expression and fluorescent tagging of α-synuclein in the gut of adult mice after tamoxifen administration.

Mice homozygous for the SNCAbow allele are viable and fertile with no abnormal phenotype. No leakiness of SNCA^WT:TagBFP, SNCA^A30P:mTFP1, or SNCA^A53T:mKO2 expression has been detected prior to Cre recombination.

Distribution of this strain was supported by the Michael J. Fox Foundation for Parkinson's Research.

Development

A targeting vector containing (from 5' to 3') a strong CAG (CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid) promoter, three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP), and the α‐synuclein-Crainbow (SNCAbow) construct (described in greater detail below), followed by an FRT-flanked neomycin resistance (neo) cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into (129S6/SvEvTac x C57BL/6NCr)F1-derived G4 embryonic stem (ES) cells and correctly targeted ES cells were microinjected into ICR/Hsd morulae. The resulting SNCAbow mice were bred with ROSA26::FLPe mice (Stock No. 003946) to remove the neo cassette and the flp-expressing allele was bred out of the colony. These mice were intercrossed for at least 3 generations by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize 129S1/SvImJ oocytes (Stock No. 002448).

Specifically, the SNCAbow construct contains three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP) that separate four cassettes, three of which are associated with a corresponding α-synuclein gene sequence. The first cassette expresses a chemically inducible near‐infrared fluorogen‐activating peptide (FAPMars1). The next three cassettes encode a unique nuclear localized fluorescent protein (TagBFP:blue, mTFP1:turquiose, or mKO2:orange) and a corresponding human α-synuclein protein sequence (SNCA^WT, SNCA^A30P, or SNCA^A53T, respectively) each separated by a P2A peptide to mediate ribosomal skipping. Each α-synuclein sequence contains a C-terminal epitope tag: SNCA^WT tagged with V5, SNCA^A30P tagged with 3XHA, or SNCA^A53T tagged with Myc. To ensure that only one fluorescent protein is expressed at any one time, an SV40 polyadenylation signal was inserted downstream of each open reading frame. Upon cre activation, the lox sites recombine to create three mutually exclusive excision possibilities with the fluorescent protein that is placed first after the CAG promoter being expressed.

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-HL9-MG*V70A,-mBFP,-SNCA,-mTFP1,-SNCA*A30P,-mKO2,-SNCA*A53T)Rali}
Name: targeted mutation 1.1, Rodger A Liddle
MGI accession ID: MGI:7332841
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence; Humanized sequence; Epitope tag
Synonyms: SNCAbow
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: A targeting vector contains (from 5' to 3') a strong CAG (CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid) promoter, three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP), and the alpha‐synuclein-Crainbow (SNCAbow) construct (described in greater detail below), followed by an FRT-flanked neomycin resistance (neo) cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. The alpha‐synuclein-Crainbow construct contains three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP) that separate four cassettes, three of which are associated with a corresponding alpha-synuclein gene sequence. The first cassette expresses a chemically inducible near‐infrared fluorogen‐activating peptide (referred to as FAPMars1). Mars1 is a V70A mutation of the human single-chain antibody (scFv)-based fluorogen isolated from HL9-MG (human library 9 malachite green). The next three cassettes encode a unique nuclear localized fluorescent protein (TagBFP:blue, mTFP1:turquoise, or mKO2:orange) and a corresponding human alpha-synuclein protein sequence (SNCA(WT), SNCA(A30P), or SNCA(A53T), respectively) each separated by a P2A peptide to mediate ribosomal skipping. Each alpha-synuclein sequence contains a C-terminal epitope tag: SNCA(WT) tagged with V5, SNCA(A30P) tagged with 3XHA, or SNCA(A53T) tagged with Myc. To ensure that only one fluorescent protein is expressed at any one time, an SV40 polyadenylation signal was inserted downstream of each open reading frame. Upon cre activation, the lox sites recombine to create three mutually exclusive excision possibilities with the fluorescent protein that is placed first after the CAG promoter being expressed. FLP-mediated recombination removed the neomycin cassette.