STOCK Ptprc^a Lag3^tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1^a/J

Stock number: 025855
Strain synonyms: Luc⁺Lag-3^-
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Mice harboring the CAG-luc-eGFP L2G85 transgene and lacking Lag3 exhibit widespread expression of firefly luciferase directed by the CAG promoter, making them useful in studies of transplantation, noninvasive lineage mapping, in vivo bioluminescence imaging and technology development. LAG3 deficiency in these mice enhances the effect of GVHD.

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). Exons 1-3 of lymphocyte-activation gene 3 (Lag3) have been replaced with a neomycin resistance gene in reverse orientation to the gene. These mice also harbor congenic Ptprc^a (Cd45.1) and Thy1^a (Thy1.1) cell surface markers allowing for the tracking of these donor cells in vitro. Lag3 gene product is a transmembrane protein expressed in stimulated T lymphocytes and activated natural killer cells. LAG-3 has been found to negatively regulate cellular proliferation, activation, and homeostasis of T cells, and plays a role in Treg suppressive function.

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. Following luciferin injection, luciferase expression is generally greater in males than females, and the emitted light is adequate to penetrate the tissues of the mice so it can be detected externally with low-light imaging cameras. When mice receive a Lag3 -/- conventional T cell transfer, imaging shows that there is a significant increase in the activation and proliferation compared to transfer of WT conventional T cells. Lag3 deficiency on CD4, but not CD8 T cells, enhances the effect of GVHD. 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 (Stock No. 008450) strain, no GFP fluorescence is detected in hematopoietic tissues by flow cytometric analysis.

In 2021-2022, advanced quality control analysis performed by The Jackson Laboratory on the FVB-congenic derivative of these CAG-luc-eGFP mice (Stock No. 008450) identified 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.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

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 backcrossed to FVB for 20 generations. Dr Robert Negrin obtained these mice and further backcrossed them to B6 for at least 16 generations. These mice also harbor congenic Ptprc^a (Cd45.1) and Thy1^a (Thy1.1) cell surface markers.

Another targeted mutation was developed that replaced exons 1-3 of lymphocyte-activation gene 3 (Lag3) with a neomycin resistance gene in reverse orientation to the gene. The construct was electroporated into 129S2/SvPas-derived D3 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric males were bred to C57BL/6 females. These mice were backcrossed at least five generations to C57BL/6 mice before being bred to the B6 CAG-luc-eGFP mice. Upon arrival at The Jackson Laboratory, mutant mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

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 on the FVB-congenic derivative of these CAG-luc-eGFP mice (Stock No. 008450) identified 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.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Two of the 27 markers throughout the genome were segregating suggesting an incomplete backcross. One marker on Chr. 7 may be due to transgene insertion. Also, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6 ; C57BL/6N genetic background.

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.

Allele

Symbol: Lag3^tm1Doi
Name: targeted mutation 1, Christophe Benoist and Diane Mathis
MGI accession ID: MGI:2176269
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Lag3
Marker name: lymphocyte-activation gene 3
Chromosome: 6
Strain of origin: 129S2/SvPas
Site of expression: Widespread.
Molecular note: A neomycin resistance gene replaced a genomic fragment containing exons 1-3. RT-PCR analysis on RNA derived from concanavalin A (Con A) induced splenocytes confirmed that no detectable transcript was produced from this allele.

Allele

Symbol: Ptprc^a
Name: a variant
MGI accession ID: MGI:4819849
Generation method: Not Applicable
Marker symbol: Ptprc
Marker name: protein tyrosine phosphatase, receptor type, C
Chromosome: 1
Strain of origin: Not Applicable
Site of expression: Widely expressed on all adaptive and innate immune cells.
Molecular note: Ptprc^a is found in strains SJL/J, STS/A, and DA. Ptprc^b is found in strains C57BL/6, C3H/An, DBA/2, AKR, and many others (J:13367, J:12054, J:12077, J:8603). Twelve nucleotide differences between the a and b alleles have been identified. These base substitutions correspond to five amino-acid changes within the extracellular domain of the encoded protein. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat sequences (J:22485).

Note that the allele designations originally described were reversed in 1987 (J:8603); all publications prior to 1987 show SJL/J, STS/A, and DA as having the b allele and the C57BL/6J group as having the a allele (J:22341).

Allele

Symbol: Thy1^a
Name: a variant
MGI accession ID: MGI:3579311
Generation method: Not Applicable
Marker symbol: Thy1
Marker name: thymus cell antigen 1, theta
Chromosome: 9
Strain of origin: Not Applicable
Site of expression: The Thy1 locus determines a surface antigen present on cells of the thymus, a number of mouse leukemias, brain, and in lesser amounts on lymph node and spleen cells.
Molecular note: The allele Thy1^a determines an antigenic specificity, Thy-1.1, found in the AKR and RF strains.
General note:

The Thy1 locus determines a surface antigen present on cells of the thymus, a number of mouse leukemias, brain, and in lesser amounts on lymph node and spleen cells. The allele Thy1^a determines an antigenic specificity, Thy-1.1, found in the AKR and RF strains; the allele Thy1^b determines an antigenic specificity, Thy-1.2, found in the C3HeB/Fe and many other strains (J:5243, J:5012, J:4469). The Thy1 antigen is probably present on all T lymphocytes and absent from all B lymphocytes, and it thus serves as a valuable T-cell marker (J:5243). It is very widely used in experiments designed to determine the distribution and function of T-cells. Thy1 specifies a T-cell surface glycoprotein, T25, with a molecular weight of 25 kDa (J:5707). The protein appears to be anchored in the cell membrane by a lipid that is either phosphotidylinositol or closely related to it (J:12016). Thy1 may function in the cell membrane as a signal transduction molecule (J:8333). The Thy1 locus, or possibly a gene closely linked to it, controls quantitative expression of a protein that is the same size as Thy1 and is expressed on thymus and brain but not on lymph node and spleen cells (J:7900).