Breeding of homozygous mice of Treg-Th17 fluorescence double labeling and their preliminary use in endotoxemia study / 中华创伤杂志

ABSTRACT

Objective To breed homozygous mice of Treg-Th17 fluorescence double labeling and to investigate the role of Treg-Th17 balance in endotoxemia. Methods Mice of Treg and Th17 fluorescence single labeling were brought from Harvard Medical School,USA and were mated and bred based on the genetic rules.The genomic DNA was extracted from the tails of second generation weaning mice for genotyping by polymerase chain reaction (PCR).Thereafter,the homozygous mice of fluorescence double labeling were selected to have successive inbreeding for three generations and their genotypes and growth status were measured.The endotoxemia model in vivo was duplicated and changes of Treg-Th17 balance were detected by flow cytometry analysis at 24 h and 48 h after endotoxemia. Results Among the seven second generation mice in the first batch,only three (two males and one female) were the wanted homozygotes.Meanwhile,the genotypes of their offsprings for the next three generations were all Foxp3 RFPKI-homo IL-17A GFPKI with normal growing status.As important component of endotoxin,lipopolysaccharide (LPS)could induce significant signal expressions of red fluorescence protein (RFP) and green fluorescence protein (GFP).The ratio of CD8a + Foxp3 +,CD8a + IL-17A +,CD4 + Foxp3 + and CD4 + IL-17A + in the lymphocytes went up significantly in the group of 24 hours of LPS treatment,as compared with the control group (P ＜ 0.01 or P ＜ 0.05 ),while the difference between the control group and the group of 48hours of LPS treatment had no statistical significance (P ＞ 0.05). Conclusion Foxp3 RFPKI-homoIL-17A GFPKI mice have identical genotypes and stable genetic characteristics and is a model animal and novel research platform that can provide real-time monitoring of the changes of Treg-Th17 balance in vivo.The role of T reg-Th17 balance works significantly at 48 hous after the subject is exposed to LPS stimulation,and the roles of CD8a + Foxp3 and CD8a + IL-17A + in Treg-Th17 balance require further investigation.