STOCK Cd8a^{tm2.1(Cd4)Asin} Cd4^{tm1(Cd8b1,Cd8a)Asin}/J

Stock number: 037806
Product name: Cd4^CD8Cd8^CD4
Strain synonyms: FlipFlop
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Cd4^CD8Cd8^CD4 knock-in/knock-out, or "FlipFlop", mice have CD4 antigen (Cd4) and CD8 antigen, alpha chain (Cd8a) gene loci that encode the opposite CD4 and CD8 coreceptor proteins of wildtype mice. These mice may be useful in studying T-cell differentiation and T-cell immunity.

As these double homozygous Cd4^CD8Cd8^CD4 mice are immunocompromised, SPF (specific pathogen-free) husbandry conditions are recommended.

Detailed description

Cell surface proteins CD4 and CD8 are expressed on T helper cells and cytotoxic T lymphocytes, respectively. These T cell co-receptors aid in differentiation of immature thymocytes and facilitate activation of the T cell receptor (TCR) and major histocompatibility (MHC) complex in the immune response pathway. To aid in the understanding of T cell lineage fate determination, the donating laboratory developed these Cd4^CD8Cd8^CD4 knock-in/knock-out, or "FlipFlop", mice whose Cd4 and Cd8a genes encode the opposite coreceptor proteins of wildtype (WT) mice, functionally reversing the T cell immune system and generating CD4-positive cytotoxic T cells and CD8-positive helper T cells.

Double homozygous Cd4^CD8Cd8^CD4 mice are viable and fertile. However, the donating laboratory has evaluated immune response in these mice and noted that the FlipFlop mice fail to promote protective immunity against environmental pathogens. After immunization with a soluble antigen, the reversed co-receptor T cells of these double homozygous mice, were unable to interact with antigen-specific B cells. It was also determined that the FlipFlop mice failed to clear a virus after acute exposure of lymphocytic choriomeningitis virus Armstrong strain (LCMV-Arm). These Cd4^CD8Cd8^CD4 mice may be useful in studying T-cell lineage fate and T-cell immunity.

As these double homozygous Cd4^CD8Cd8^CD4 mice are immunocompromised, SPF (specific pathogen-free) husbandry conditions are recommended. The donating investigator notes that the floxed neomycin selection cassette was not removed from the Cd4^{tm1(Cd8b1,Cd8a)Asin} allele as the excision of the cassette reduced protein expression.

Development

The Cd4^CD8Cd8^CD4 knock-in/knock-out mice (also called "FlipFlop") harbor two targeted mutations, each described below.

The Cd8a^{tm2.1(Cd4)Asin} allele (called Cd8^CD4 or 4in8) was generated by replacing exons 1-4 of the CD8 antigen, alpha chain (Cd8a) gene on chromosome 6 (~71.6 Mb; GRCm39) with CD4 antigen (Cd4) cDNA and a floxed neomycin selection cassette. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and the resulting chimeric males were bred to EIIa-Cre transgenic females to remove the neo cassette. The resulting colony of Cd8^CD4/+ mice, with the neo cassette removed, were maintained on a 129 background by the donating laboratory (see SNP note below).

The Cd4^{tm1(Cd8b1,Cd8a)Asin} allele (called Cd4^CD8 or 8in4) was generated by inserting a targeting vector into the translation start site in exon 2 of the Cd4 gene on chromosome 6 (~124.9 Mb; GRCm39). The target construct consisted of a TK cassette, Cd8b cDNA, IRES, Cd8a1 cDNA, a polyA sequence and floxed neomycin selection cassette. The construct was electroporated into C57BL/6-derived embryonic stem (ES) cell and correctly targeted ES cells were injected into recipient blastocysts. The resulting Cd4^CD8 mice, with floxed neo cassette intact, were crossed to Cd8^CD4 mice to produce the Cd4^CD8Cd8^CD4 double mutant. The donating investigator notes that the floxed neomycin selection cassette was not removed from the Cd4^CD8 allele as the excision of the cassette reduced protein expression.

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

In 2023, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 males sent to The Jackson Laboratory Repository. Two of the 43 markers throughout the genome were segregating with an unknown source, on chromosomes 9 and 13. Also, 5 markers were segregating with 129. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed genetic background.

Allele

Symbol: Cd4^{tm1(Cd8b1,Cd8a)Asin}
Name: targeted mutation 1, Alfred Singer
MGI accession ID: MGI:7388565
Generation method: Targeted
Attributes: Null/Knockout; Inserted expressed sequence
Synonyms: 8in4; Cd4(CD8)
Marker symbol: Cd4
Marker name: CD4 antigen
Marker synonyms: CD4mut; IMD79; L3T4; Leu-3; Ly-4; OKT4D; p55; T4; W3/25
Chromosome: 6
Strain of origin: C57BL/6
Molecular note: The allele was generated by inserting a targeting vector into the translation start site in exon 2 of the gene. The target construct consisted of a TK cassette, Cd8b1 cDNA, IRES, Cd8a cDNA, a polyA sequence and floxed neomycin selection cassette.

Allele

Symbol: Cd8a^{tm2.1(Cd4)Asin}
Name: targeted mutation 2.1, Alfred Singer
MGI accession ID: MGI:5309010
Generation method: Targeted
Attributes: Null/Knockout; Inserted expressed sequence
Synonyms: 4in8; Cd8a^CD4
Marker symbol: Cd8a
Marker name: CD8 antigen, alpha chain
Marker synonyms: CD8; CD8alpha; IMD116; Leu2; Ly-2; Ly-35; Ly-B; Lyt-2; p32
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: Exons 1-4 were replaced with a Cd4 cDNA and a floxed neomycin selection cassette. The selection cassette was subsequently removed by cre excision.