After incubating with Quick-block buffer (Beyotime, Shanghai, China) for 45?min, the sections were incubated with the rabbit antichicken Sox2 antibody or commercial rabbit antimouse Sox2 antibody at 4C overnight

After incubating with Quick-block buffer (Beyotime, Shanghai, China) for 45?min, the sections were incubated with the rabbit antichicken Sox2 antibody or commercial rabbit antimouse Sox2 antibody at 4C overnight. spermatogonia in the seminiferous tubules, and a small number of Sertoli and Leydig cells, were positive for Sox2. The antichicken Sox2 antibody was also successfully used to investigate the expression and distribution of Sox2 in the chicken cerebellar cortex, optic tectum, cerebral cortex, and lungs. The results of this study confirmed the specificity of the antichicken Sox2 polyclonal antibody, which will be available for the study of biological functions of the chicken gene and the self-renewal mechanisms of chicken pluripotent stem cells. Key words: prokaryotic expression, Sox2 antibody, specificity, histochemistry, chicken Introduction As an important transcription factor, Sox2 is involved in embryogenesis, the maintenance of stem cells, and proliferation of primordial germ cells (Arnold et?al., 2011). Sox2 has been reported to be necessary for primordial germ cell proliferation and pluripotency in mice (Huangfu et?al., 2008, Nagamatsu et?al., 2013, Nettersheim et?al., 2016). Furthermore, chicken feather follicle cells in?vitro have been induced to form pluripotent stem cells through ectopic expression of the transcription BMH-21 factors, Sox2, Oct4, c-Myc, and Klf4 (Kim et?al., 2017). Lately, with the addition of Lin28 and Nanog to the aforementioned 4 transcription factors, adult chicken fibroblast BMH-21 cells were reprogrammed into pluripotent stem cells using the same methods (Katayama et?al., 2018). Furthermore, Sox2 is usually expressed in numerous adult endodermal and ectodermal stem cell compartments and is critical for normal tissue regeneration and survival (Arnold et?al., 2011). Sox2 expression in the brain was found to significantly decrease with ageing in both humans and rodents, suggesting that this decline in Sox2 expression could be used as a biomarker of ageing (Carrasco-Garcia et?al., 2019). In addition, Sox2 has been identified as a diagnostic marker for embryonic carcinoma and seminoma (Nonaka, 2009). However, little is known about the expression and distribution of Sox2 in adult chicken tissues, such as testes, brain, and lungs. Anti-Sox2 antibodies are indispensable for studies into the biological functions of Sox2 and the self-renewing mechanism of stem cells. However, the anti-Sox2 antibodies currently available are antihuman/antimouse Sox2, which may be not be highly specific for the detection of Sox2 expression in chicken cells and tissues. In this study, the chicken gene was amplified by RT-PCR from your testis of a newly hatched rooster, and the prokaryotic plasmid pET-was constructed. The His-Sox2 fusion protein expression was induced in BL21 (DE3), and the protein was purified by affinity chromatography. The purified protein was subsequently used as an antigen to prepare the antichicken Sox2 antibody by immunizing New Zealand white rabbits. Finally, the specificity of the antibody was analyzed, and the expression and distribution of Sox2 in the tissues of testes, lungs, and brain was investigated by western blotting and immunohistochemistry. Materials and methods Experimental Animals The use of all animals in the present study was approved and permitted by the Committee of Animal Welfare, Guangxi University or college, China. Plasmid Construction A pair of primers (forward 5-ATGGATCCATGTACAACATGATGGC-3 and reverse 5-CCGCTCGAGTCACATATGTGATAGAG-3, with the 2 2 underlined parts indicating mRNA sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”U12532″,”term_id”:”595494″,”term_text”:”U12532″U12532) in GenBank. The chicken gene was amplified with Nid1 RT-PCR from your testis of a 20-day-old cockerel and, subsequently, cloned into a pMD18?T vector (Takara, Japan) to construct pMD18T-by TA cloning. After DNA sequence analysis and online signal peptide prediction, pMD18T-was digested by gene was subcloned to a pET30a vector (Novagen, Germany) to construct the recombinant plasmid pET-PCR product was ligated into a pENTR/D-TOPO vector (Invitrogen) to generate the pENTR/D-TOPO-entry vector, which was subsequently ligated to pLenti6.4/R4R2/V5-DEST to create the pLenti6.4-expression vector by a MultiSite Gateway LR recombination reaction. For the CRISPR/Cas9 effector system (dCas9-KRAB)-mediated repression assay, the sgRNA for was designed using an online tool (https://zlab.bio/guide-design-resources), and its forward 5-ACACCGCCCCCCAGCAAACTTCGGGG-3 and reverse 5-AAAACCCCGAAGTTTGCTGGGGGGCG-3 oligonucleotides (underlined parts are overhang sequences) were synthesized. After annealing, the DNA oligonucleotides were cloned into the sgRNA Puro. Prokaryotic Expression and Purification of His-Sox2 Fusion Protein Expression of the His-Sox2 fusion protein by BL21 (DE3) cells was conducted as previously described (Duan et?al., 2019). For purification of the His-Sox2 fusion protein, 500?mL of Luria-Bertani media was inoculated with 5-mL overnight culture of BL21 (DE3), BMH-21 which was previously transformed with the pET-plasmid and allowed to grow to an optical density (600?nm?=?0.6C0.8) by incubation at 37C with vigorous shaking. The His-Sox2 fusion protein was subsequently induced for 4?h by addition of 1-mmol isopropyl -d-1-thiogalactopyranoside. Cells were then pelleted, washed once with phosphate buffered saline (PBS), and resuspended in buffer B (100?mmol NaH2PO4, 10?mmol TrisCl, 8?mol urea, pH 8.0), followed by incubation for 1?h?at room temperature with gentle shaking. This suspension was sonicated on ice for 20?min and pelleted for 20?min?at 13,000?g. The supernatant was collected and filtered through a 0.45-m syringe filter. The cleared lysate was slowly loaded into a packed column with nickel-nitrilotriacetic acid metal-affinity chromatography matrices (Qiagen, Dusseldorf, Germany) for washing nonspecific.