Phenotypic Analysis Of Tmprss11f Knockout Mice

Objective:Type ? transmembrane serine proteases (TTSPs) are a group of proteolytic enzymes of the trypsin superfamily. Structurally, all TTSPs contain a transmembrane domain near the N-terminus and an extracellular protease domain at the C-terminus. The transmembrane domain anchors the TTSPs on the cell surface, whereas the protease domain mediates proteolytic catalysis. TTSP family members include enteropeptidase, hepsin, corin, matriptases and TMPRSSs, which are involved in a variety of biological and pathological processes, such as food digestion, cancer progression, blood pressure regulation, iron metabolism and normal hearing. Defects in TTSPs may lead to malnutrition, hypertension, iron-deficiency anemia and recessive non-syndromic deafness. Human TTSPs include 19 members, which can be further divided into four subgroups, i.e. hepsin/TMPRSS, matriptase, HAT/DESC and corin subgroups.TMPRSS11f, also called HAT-Like 4 (HATL4), is a TTSP of the HAT/DESC subgroup. Most recent studies in our lab indicated that TMPRSS11f was not expressed in normal blood and bone marrow cells but abnormally up-regulated in acute myeloid leukemia (AML) cancer cells. In addition, the abnormal TMPRSS11f expression was associated with poor clinical outcomes in AML patients, suggesting that TMPRSS11f may contribute to the pathogenesis of AML. To date, however, little is known regarding TMPRSS11f expression in human and mouse tissues and its normal biological functions.The goal of this study is to examine TMPRSS11f expression in human and mouse tissues and to analyze the phenotype of Tmprss11f knockout mice, which were created by CRISPR/Cas9-based technology. We designed experiments to analyze the viability, postnatal growth, fertility, and long-term survival of Tmprss11f knockout mice. We also planned studies to analyze blood cells and chemistry and to test experimental models for wound healing and skin function in the knockout mice.Methods:1) Reverse transcription PCR (RT-PCR) was used to analyze TMPRSS11f mRNA expression in mouse tissues.2) Immunohistochemistry was used to analyze TMPRSS11f protein expression in human and mouse tissues.3) CRISPR/Cas9-based technology was used to delete exon 4 of the Tmprss11f gene to create Tmprss11f knockout mice.4) PCR and RT-PCR were used to genotype Tmprss11f knockout mice and to verify the lack of Tmprss11f mRNA expression in the knockout mice.5) Offspring from Tmprss11f+/- mating was genotyped to determine the Mendelian distribution ratio.6) Litter size and offspring body weight at birth and weaning from the Tmprss11f+/-mating were examined.7) Body weight gain, fertility and long-term survival of Tmprss 11 f knockout mice were analyzed.8) Blood cell and chemistry analysis was performed in Tmprss11f knockout mice.9) Wound healing and swimming tests were conducted to analyze the skin function in Tmprss11f knockout mice.Results:1) TMPRSS11f protein was detected in human tissues, including skin, trachea, esophagus, vagina, placenta, testis, and ovary. In mice, TMPRSS11f mRNA and protein were detected in eye, trachea, tongue, esophagus, ureter, bladder, seminal vesicle, ductus deferens, placenta, corpora uteri, cervix, vagina and skin.2) In the trachea, TMPRSS11f protein was located in cilia and glands, whereas in the skin TMPRSS11f protein was expressed in sebaceous glands.3) Tmprss11f knockout mice were created and verified by genotyping and mRNA analysis.4) Mating of Tmprss11f+/-mice produced offspring with an expected Mendelian ratio.5) Tmprss11f-/- mice were viable and fertile and exhibited similar growth and long-term survival to that in wild-type mice.6) Blood cell and chemistry analysis did not detect significant differences between Tmprss11f-/- and wild-type mice.7) No apparent abnormalities were found in Tmprss11f-/- mice by histological analysis of major organs.8) In experimental models, Tmprss11f-/- and wild-type mice had similar responses in wound healing and skin-dependent thermoregulation.Conclusions:By RT-PCR and immunohistochemistry analysis, we found that TMPRSS11f is widely expressed in epithelial tissues, including eye, trachea, tongue, esophagus, ureter, bladder, seminal vesicle, ductus deferens, placenta, corpora uteri, cervix, vaginal vault and skin.We created Tmprss11f knockout mice using CRISPR/Cas9-based technology. Phenotypic analysis of Tmprss11f-/- mice indicates that TMPRSS11f is dispensable for embryonic development, postnatal growth and long-term survival. Analysis of blood cell and chemistry did not found apparent defects in Tmprss11f-/- mice. In experimental models, Tmprss11f-/- and wild-type mice had similar responses in wound healing and skin-dependent thermoregulation. Our results suggest that TMPRSS11f may play a regulatory role in epithelial function that is only exhibited upon additional challenges. The knockout mice we created should be a useful model for further studies of TMPRSS11f function in physiological and pathological processes.