FVB-Tg(CAG-luc,-GFP)L2G85Chco/J

Stock number: 008450
Product name: L2G85
Research areas: Research Tools

Description

Mice harboring the CAG-luc-eGFP L2G85 transgene exhibit expression of firefly luciferase directed by the CAG promoter (human cytomegalovirus immediate early promoter enhancer with chicken beta-actin/rabbit beta-globin hybrid promoter). These CAG-luc-eGFP L2G85 transgenic mice may be useful in studies of transplantation, noninvasive lineage mapping, in vivo bioluminescence imaging and technology development.

The transgene integration site was identified to be on chromosome 7 in exon 10 of the Ptprh locus and likely results in a Ptprh knock-out allele [see Detailed Description section for more information].

Detailed description

Mice homozygous for the CAG-luc-eGFP L2G85 transgene are viable and fertile, with expression of firefly luciferase and enhanced green fluorescence protein directed by the CAG promoter (human cytomegalovirus immediate early promoter enhancer with chicken beta-actin/rabbit beta-globin hybrid promoter). Bioluminesence is detected in heart, spleen, muscle, pancreas, skin, thymus and bone marrow. Luciferase activity is not detected in mature erythrocytes, although low levels are detected in erythrocyte precursors and varying levels of activity in all leukocyte subsets tested. Homozygotes have no reported gross physical or behavioral abnormalities. Following luciferin injection, luciferase expression is generally greater in males than females. GFP fluorescence is detected in skin (upper epidermal layers) by fluorescence microscopy. The Donating Investigator reports that for the FVB-Tg(CAG-luc,-GFP)L2G85Chco/J strain (Stock No. 008450), no GFP fluorescence is detected in hematopoietic tissues by flow cytometric analysis.

In 2021-2022, advanced quality control analysis performed by The Jackson Laboratory identified Stock No. 008450 to have the CAG-luc-eGFP transgene inserted into Ptprh exon 10 [Chr7:4,558,956-4,569,937 ; mm10]. Luciferase ddPCR performed on homozygous mice (n=17) showed ~11-19 copies of the transgene. Western blot of intestinal tissue from homozygous mice (n=6) showed no PTPRH expression, suggesting the transgene insertion results in a Ptprh knock-out allele.

Development

The CAG-luc-eGFP transgenic construct was designed by the laboratory of Dr. Christopher H. Contag (Stanford University School of Medicine). Specifically, the transgene contained the human cytomegalovirus immediate early promoter enhancer with chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from the pCAGGS vector) upstream of a modified firefly luciferase gene open reading frame, 54 bp of the foot and mouth disease virus (FMDV) 2A sequence (ribsome slippage site), 24 bp polylinker, and enhanced Green Fluorescent Protein (eGFP) open reading frame. This transgene was injected into fertilized FVB mouse eggs. Founder line L2G85 animals were bred to wildtype FVB mice. These FVB-L2G85 (or FVB.luc) transgenic were then backcrossed to FVB for 20 generations prior to arrival at The Jackson Laboratory in 2009. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize FVB/NJ oocytes (Stock No. 001800). The colony was maintained by breeding transgenic mice together, with additional FVB/NJ backcrosses occurring in 2015 and 2019. In 2021, we recovered mice from the 2009 frozen sperm (fertilizing FVB/NJ oocytes) and compared homozygous transgene copy number to the existing homozygous live colony. Both lineages showed similar copy number (see more information below). By January 2022, the live colony was replaced by the 2021 recovered lineage.

In 2011, fine mapping performed at The Jackson Laboratory on the NOD-congenic derivative of these CAG-luc-eGFP mice (Stock No. 010542) indicated that the transgene integrated onto the proximal ~37 Mb of Chromosome 7 (hemizygous mice showed the 5 most proximal Chromosome 7 markers in the analysis - D7Mit306 [~10 Mb] through D7Jmp8 [~29 Mb] - all had contributions from FVB).

In 2021-2022, advanced quality control analysis performed by The Jackson Laboratory identified Stock No. 008450 to have the CAG-luc-eGFP transgene inserted into Ptprh exon 10 [Chr7:4,558,956-4,569,937 ; mm10]. Luciferase ddPCR performed on homozygous mice (n=17) showed ~11-19 copies of the transgene. Western blot of intestinal tissue from homozygous mice (n=6) showed no PTPRH expression, suggesting the transgene insertion results in a Ptprh knock-out allele.

Allele

Symbol: Tg(CAG-luc,-GFP)L2G85Chco
Name: transgene insertion L2G85, Christopher H Contag
MGI accession ID: MGI:3844218
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(CAG-luc,-GFP)L2G85Chco
Marker name: transgene insertion L2G85, Christopher H Contag
Chromosome: 7
Strain of origin: FVB
Expressed genes: luc,luciferase,firefly; GFP,Green Fluorescent Protein,
Site of expression: Widespread expression of firefly luciferase is reported in all tissues and organs, except mature erythrocytes. Following luciferin injection, luciferase expression is generally greater in males than females. GFP fluorescence is detected in skin.
Molecular note: A transgenic construct containing a modified firefly luciferase gene open reading frame, 54bp of the foot and mouth disease virus (FMDV) 2A sequence (ribsome slippage site) and eGFP sequence under the control of the chicken beta actin promoter coupled with the cytomegalovirus (CMV) immediate early enhancer (CAG), was injected into fertilized FVB mouse eggs. Founder line L2G85 animals were bred to wildtype FVB mice.
General note: Fine mapping at The Jackson Laboratory indicates that the transgene inserted into Ptprh exon 10 [Chr7:4,558,956-4,569,937 ; mm10]. Luciferase ddPCR performed on homozygous mice (n=17) showed ~11-19 copies of the transgene. Western blot of intestinal tissue from homozygous mice (n=6) showed no PTPRH expression.