Genetic Models Induced By ENU Mutagenesis For T Lymphocyte Development Research

BackgroundENU mutagenesis is a powerful approach to identify novel genes or alleles to dissect the molecular basis of mammalian immune system.T cells are critical components in the host defense against pathogens and infection,CD4 is a co-receptor for T cell activation,and structural studies showed that multiple extra-cellular domains of CD4 exist on T cell surface,including the first extracellular domain that interacts with its ligand MHCII.However functional studies elucidating how the extracellular domain maintain its interaction with MHCII,on the basis of in vivo mouse models,are still limited.ObjectiveIn this study,we created immunization-deficient mouse model in C57BL/6J strain using ENU mutagenesis,investigate the molecular mechanism of deficiency of CD4 helper T cell and the critical role of the first extra-cellular domain in vivo.It could be an optimal model for study of CD4 helper T cells.Methods1.Thirty male C57BL/6J mice(G0)aged 8 weeks were injected with three dose of90mg/kg ENU(Sigma,N-3385)intraperitoneally once a week for three consecutive weeks.7 weeks after the last injection,each male mice was crossed to one C57BL/6J female mouse to produce the first generation progeny(G1).Brother-sister mating of G1 mice were performed to produce G2 animals,which were then intercrossed to produce G3 mice for phenotyping.2.Around 100 ?l peripheral blood of G3 mice were collected with anticoagulants.30?l blood was stained with 10?l anti-mouse antibodies mix.400 ?l BD FACS? lysing solution dilluted by distilled water was used for lysing erythrocytes at room temperature for 10 minutes.Cells were analysed with flow cytometry BD FACS Canto?.3.We designed the mutant Cd4 gene sequence and cloned it into the mammalian expression vector pCI-neo,yielded an intermediate plasmid pCI-mCD4.A fragment containing T2A-EGFP was cloned into the same cloning site of vector pCI-mCD4.The final construct was named pCI-mCD4-EGFP.4.HEK293 T cells were transfected with plasmid pCI-mCD4-EGFP and pCI-mCD4 respectively along with a no-transduction control.Cells were incubated at 37? for 24~72hours and prepared for FACS analysis.5.Mouse with knockin alleles of Ly5.1 Foxp3-eGFP were used as donor mice,CD4 T cells were purified from the secondary lymph nodes cells and spleencytes and purified by Dyna-beads? Untouched ? Mouse CD4 Cells Kit.The mutant mice were used as the recipients and CD3 e knockout mice were used for comparison.Each recipient mouse was retro-orbitally injected CD4 T cells following purification.6 weeks after transfer,blood cells and spleenocytes were analyzed for T cell frequencies including the regulatory T cell frequency by flow cytometry.Results1.A mutant line was found to be deficient in CD4 T cell compartment.In blood,the other lineages of leukocytes were found normal in frequency distribution,but the CD4 T cell subset was completely absent in G3 mice.As progeny from crosses between phenotypic mutant mice and wild type mice had normal CD4 T cell distribution,and G3 animals showed Mendelian ratio of mutant phenotype,the mutation was recessive and of autosomal inheritance.2.The mutation caused by ENU mutagen was identified as a T to A point mutation of124873055 in Cd4 gene on Chr 6 by exome capture and next generation sequencing.And the consequence of the mutation was a non-synonymous coding change from isoleucine to asparagine at the position of 99 of CD4.3.The isoleucine to asparagine mutation in CD4 extra-cellular domain completely abolish its function in T cell development.4.The isoleucine to asparagine mutation at the position of 99 of CD4 does not affect its surface expression.5.The ENU induced CD4 deficient mouse in C57BL/6J background is a desirable model for studies involving CD4 T cell transfer.ConclusionsENU mutagenesis resulted in a point mutation of T?A at 124873055 site of Cd4 gene on Chr 6,whereby induced a loss of function mutation from Ile to Asn at the position 99 of CD4,gave rise to a mutant mouse line that was absent completely in CD4 helper T lyphocytes.The mutant mice were an optimal recipient model to perform CD4 T cell adoptive transfer experiments.