Anti-FLAG tag antibody, clone OZSFLAG

Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flags) In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.

Argonaute (AGO) family proteins, which are key components of RNA-induced silencing complexes (RISCs), associate with both microRNAs (miRNAs) and mRNAs to regulate gene expression at post-transcriptional stages. While high-quality antibodies for AGO proteins are extremely useful for studies on the functions of miRNAs and AGO proteins, they are not always readily available. In this study, we generated a knock-in human embryonic stem cell (hESC) line by using homologous recombination to express FLAG-tagged AGO2 from its native genomic locus. FLAG-tagged AGO2 was exploited to analyze AGO2-bound miRNAs in hESCs cultured on a feeder layer by immunoprecipitation. Furthermore, the pluripotency of ESCs allowed for the analysis of miRNAs after differentiation into fibroblasts and cardiomyocytes. The genetically modified hESC line generated in this study will be highly useful for studying the functions of miRNA-mediated regulation in human development and cell differentiation.