Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development / 第三军医大学学报

Objective To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique.Methods gRNA was designed and prepared for the faf1 gene of zebrafish,and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos.The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing.The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish,and the genotype of zebrafish was detected by microscopic observation.Results The faf1 gRNA and Cas9 mRNA were successfully prepared.The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations.The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish.At 4 d post fertilization (dpf),there were sarcomeric dysplasia and head shrinkage,increased hyoid angle and other craniofacial cartilage deformities.And the zebrafish died at 8 ～9 dpf.Conclusion CRISPR/Cas9 knocking out thefaf1 gene produces a new phenotype for zebrafish,with delayed pigment deposition and nodule-like change in tail muscle section.

Expression of Fas-associated Factor 1 in the Developing Testis / 체질인류학회지

FAS associated factor 1 (FAF1) is a Fas-associating molecule, which enhances Fas mediated apoptosis. FAF1 gene is expressed most abundantly in the testis among the mouse organs. The aim of this study was to reveal the expression and the role of FAF1 in the developing testis. H-E stain and FAF1 immunohistochemistry were performed in the testis and epididymis of the E15.5 embryo, and 1, 2, and 8 week-old C57/BL6 mice. FAF1 was expressed in the testis from E 15.5 embryo to 8 week-old mice. Cell type of FAF1 positive cells was different among the developmental stage. Furthermore, cellular (cytoplasmic or nuclear) localization of FAF1 in the male germ cells was different during the developmental stage. FAF1 was expressed mainly in the nuclei of the germ cells 1 and 8 weeks after birth, when cell differentiation occurs actively in the testis. However, FAF1 was expressed in the cytoplasms of germ cells 2 weeks after birth, when apoptosis occurs maximally in the testis. Taken together, it can be suggested FAF1 expressed in male germ cells in the testis. FAF1 might be involved in regulation of the cellular function during spermatogenic cell differentiation and apoptosis in the testis.