STOCK Cx3cr1^tm1Litt Tg(Prism)1989Htz/J

Stock number: 031478
Product name: PrismPlus (Cx3cr1-EGFP ; Prism JD1989)
Research areas: Neurobiology Research, Research Tools

Description

PrismPlus is a quadruple-colored mouse line with EGFP fluorescence in microglia, YFP fluorescence in neurons, Cerulean fluorescence in oligodendrocytes and DsRedMax fluorescence in astrocytes. These mice may be useful for noninvasive and/or in vivo imaging of CNS neural circuitry, as well for transplantation studies or as a source for cell culture experiments.

Detailed description

PrismPlus is a quadruple-colored mouse line with EGFP fluorescence in microglia, YFP fluorescence in neurons, Cerulean fluorescence in oligodendrocytes and DsRedMax fluorescence in astrocytes.

The PrismPlus colony (Stock No. 031478) was created in 2018 by breeding C57BL/6-congenic Cx3cr1-EGFP mice (Stock No. 005582) with FVB-congenic Prism JD1989 mice (Stock No. 018068). The phenotype of the individual parental mouse lines is described further below.

The laboratory of Dr. Kevin J. Otto (University of Florida) independently assembled their own PrismPlus colony (B6;FVB genetic background) and characterized them in their PrismPlus Neuroscience 2017 poster and Gaire et_al. 2018_Sci Rep. publication. They report mice heterozygous for Cx3cr1-EGFP and hemizygous for Prism JD1989 are viable and fertile, but when bred together have small litter sizes. Additionally, they describe that breeding is enhanced when provided a high-fat diet and mouse huts. They observed no expression differences compared to that originally described for the individual parental mouse lines.

The Cx3cr1-EGFP knock-in/knock-out allele (Cx3cr1^tm1Litt; Stock No. 005582) expresses EGFP fluorescence in brain microglia, as well as monocytes, dendritic cells and NK cells - mimicking endogenous gene expression. The same subset of peripheral blood cells isolated from heterozygotes express detectable levels of EGFP. As described for Stock No. 005582, mice homozygous for Cx3cr1-EGFP are viable and fertile, normal in size and do not display any gross physical or behavioral abnormalities.

Prism JD1989 (Tg(Prism)1989Htz ; Stock No. 018068) has three co-injected BAC transgene constructs, Snap25::YFP-HA-L10a, Mobp::Cerulean-Myc and Aldh1L1::DsRedMax, integrated into the genome as multiple transgene copies at a single locus.
The Snap25::YFP-HA-L10a transgene expresses YFP fluorescence in neurons. The L10a tag restricts most of the YFP to the cell body/cytoplasm and nucleolus. YFP fluorescence is very bright in glia, and dimmer in neurons. The HA tag allows this YFP to be distinguished from other fluorophores via antibody staining.
The Mobp::Cerulean-Myc transgene expresses Cerulean fluorescence in oligodendrocytes. Cerulean has been engineered to be ~2.5-fold brighter than ECFP and have similar spectral properties (excitation maxima ~433 nm and emission maxima ~475 nm). The Myc tag allows this Cerulean to be distinguished from other fluorophores via antibody staining.
The Aldh1L1::DsRedMax transgene expresses DsRed-Max fluorescence in astrocytes. DsRed-Max is a tetramer engineered to have reduced aggregation, high solubility and bright/vivid red fluorescence (excitation maxima ~560 nm and emission maxima ~589 nm) - making it useful as a marker for directly visualizing labeled cells. Of note, this transgene expresses red fluorescence that is visible through the skull of newborn pups.
As described for Stock No. 018068, mice hemizygous for Prism JD1989 are viable and fertile, but only ~half of the expected transgenic offspring survive the early postnatal period, and survivors exhibit lower birth weight. Some transgenic mice develop lesions near the eyes. Transgene expression, for each transgenic construct, mimics endogenous gene expression patterns.

Development

PrismPlus mice (Stock No. 031478) were created in 2018 by breeding C57BL/6-congenic Cx3cr1-EGFP mice (Stock No. 005582) with FVB-congenic Prism JD1989 mice (Stock No. 018068). The Cx3cr1-EGFP allele and Prism JD1989 transgenes are described below.

The Cx3cr1-EGFP knock-in/knock-out allele (Cx3cr1^tm1Litt) was created in the laboratory of Dr. Dan R. Littman (New York University Medical Center) to have an enhanced green fluorescent protein (EGFP) cDNA sequence followed by an SV40 polyadenylation sequence replacing the first 390 bp of exon 2 of the chemokine (C-X3-C motif) receptor 1 locus (Cx3cr1) on chromosome 9. C57BL/6-congenic Cx3cr1-EGFP mice from Stock No. 005582 were used for creating the PrismPlus strain.

The Prism JD1989 transgenic mouse line (Tg(Prism)1989Htz) was created in the laboratory of Dr. Nathaniel Heintz (The Rockefeller University) by co-injection of three bacterial artificial chromosome (BAC) transgene constructs: Snap25::YFP-HA-L10a, Mobp::Cerulean-Myc and Aldh1L1::DsRedMax.
To create the Snap25::YFP-HA-L10a transgene, the BAC RP23-290A18 was obtained and modified by inserting a yellow fluorescent protein (YFP) with a C-terminal hemagglutinin tag (HA) and ribosomal protein L10A tag (Rpl10a) into the synaptosomal-associated protein 25 (Snap25) translation start site.
To create the Mobp::Cerulean-Myc transgene, the BAC RP23-172H7 was obtained and modified by inserting a Cerulean fluorescent protein with a C-terminal Myc tag into the myelin-associated oligodendrocytic basic protein (Mobp) translation start site. Cerulean is a derivative of Aequorea victoria green fluorescent protein that has been engineered to be ~2.5-fold brighter than enhanced cyan fluorescent protein (ECFP). Cerulean has excitation maxima ~433 nm and emission maxima ~475 nm.
To create the Aldh1L1::DsRedMax transgene, the BAC RP23-7M9 was obtained and modified by inserting a DsRed-Max fluorescent protein into the aldehyde dehydrogenase 1 family, member L1 (Aldh1l1) translation start site. The Discosoma sp. red fluorescent protein (DsRed) variant DsRed-Max is a tetramer with reduced aggregation, high solubility and bright/vivid red fluorescence (excitation maxima ~560 nm and emission maxima ~589 nm).
The original Prism JD1989 publication (Dougherty et al. 2012 PLoS One 7(7):e40511) suggests multiple transgene copies integrated into the genome at a single locus. The FVB-congenic Prism JD1989 mice from Stock No. 018068 were used for creating the PrismPlus strain.

Allele

Symbol: Cx3cr1^tm1Litt
Name: targeted mutation 1, Dan R Littman
MGI accession ID: MGI:2670351
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: CX3CR1^-; CX3CR1^EGFP; Cx3cr1^gfp; CX3CR1-EGFP; CX3CR1-GFP; CX3CR-GFP
Marker symbol: Cx3cr1
Marker name: chemokine (C-X3-C motif) receptor 1
Marker synonyms: CCRL1; CMKBRL1; CMKDR1; GPR13; GPRV28; Rbs11; V28
Chromosome: 9
Strain of origin: 129P2/OlaHsd
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: GFP labels T and B cells in the lymphoid organs such as spleen, small intestine, and large intestine.
Molecular note: The endogenous locus was disrupted by the insertion of sequence encoding green fluourescent protein (GFP), replacing the first 390 bp of the only coding exon. The deleted region encoded an amino-terminal portion of the protein that is crucial for interaction with endogenous ligand, Cx3cl1. A floxed neo gene included in the targeting vector for selection was excised prior to germline transmission, leaving a single loxP site downstream of the GFP sequence. RT-PCR and flow cytometry indicated an absence of endogenous protein and the presence GFP expression in homozygous mutant mice.

Allele

Symbol: Tg(Prism)1989Htz
Name: transgene insertion 1989, Nathaniel Heintz
MGI accession ID: MGI:5317680
Generation method: Transgenic
Attributes: Reporter
Synonyms: Prism JD1989; Snap25::Yfp-Rpl10a, Mobp::Cfp-myc, Aldh1L1::DsrRedMAx; Tg(Snap25-YFP/Rpl10a,Mobp-CFP,Aldh1l1-DsRedMax)1989Htz
Marker symbol: Tg(Prism)1989Htz
Marker name: transgene insertion 1989, Nathaniel Heintz
Chromosome: UN
Strain of origin: FVB
Expressed genes: CFP,Cyan Fluorescent Protein,jellyfish; RFP,Red Fluorescent Protein,coral; YFP,Yellow Fluorescent Protein,jellyfish; myc tag,myc tag,
Site of expression: Myc tagged CFP is expressed in oligodendrocytes, DsRedMax is expressed in astrocytes, and Rpl10a tagged YFP is expressed in neurons.
Molecular note: Three mouse bacterial artificial chromosome (BAC) transgenic constructs were co-injected to generate this Prism 1989 strain. The BAC RP23-290A18, containing the entire Snap25 gene, was modified by inserting Rpl10a (ribosomal protein L10A) tagged YFP into the translation start site (ATG) of Snap25. The BAC RP23-172H7, containing the entire Mobp gene, was modified by inserting a Myc-tagged Cerulean (CFP) into the translation start site (ATG) of Mobp. The BAC RP23-7M9, containing the entire Aldh1l1 gene, was modified by inserting DsRedMax into the translation start site (ATG) of Aldh1l1. Founder line 1989 was subsequently established.