| Transcription factor OCT4 serves as a critical marker during the early embryonic development of mammals,especially in pluripotent stem cells(PSCs).It plays a vital role as a regulatory gene influencing embryonic development.In PSC research,OCT4 protein expression levels are commonly monitored to evaluate the establishment and maintenance of pluripotency.In this study,we employed CRISPR/Cas9-mediated homologous recombination and electroporation techniques to construct a transgenic cell line harboring a dual reporter system consisting of OCT4-EGFP-Puro R.The objective was to precisely monitor the expression of endogenous OCT4 by driving the expression of three proteins using the endogenous OCT4 promoter.This approach will facilitate the generation of genetically modified cloned pigs with functional OCT4-EGFP-Puro R reporters,thereby providing valuable tools to advance research on early embryonic development and stem cells in pigs.Methods: In this study,Bama pig fetal fibroblasts were isolated using physical fragmentation and enzymatic digestion.The gender of the isolated pig fetuses was determined by amplifying the Y-chromosome-specific SRY gene,and the cell types were identified through cell immunofluorescence staining.Puro R-P2A-m Cherry m RNA was prepared using molecular cloning and in vitro transcription techniques,and the functionality of the m RNA resistance selection system was validated through cell screening.sgRNAs were designed,transcribed in vitro,and incubated with Cas9 protein.Gel electrophoresis and genomic DNA sequencing were performed to analyze the edited cells.The efficiency of both extracellular and intracellular cleavage by different sgRNAs was evaluated using grayscale analysis and the TIDE software.Donor DNA vectors were constructed using molecular cloning techniques,and the Donor DNA vectors,along with Cas9/sgRNA ribonucleoprotein complexes and m RNA,were delivered into Bama pig fetal fibroblasts through Lonza electroporation using CRISPR/Cas9 technology.Monoclonal cells were screened and identified through puromycin selection,PCR amplification of the target sequence with integration across homology arms,and DNA sequencing.Results: Agarose gel electrophoresis analysis revealed that that all four Bama pig fetuses in the first batch carried the SRY gene.Among the seven Bama pig fetuses in the second batch,E2-3,E2-4,and E2-5 did not have the SRY gene,whereas E2-1,E2-2,E2-6,and E2-7 did contain the SRY gene.Immunofluorescence staining of cells confirmed the expression of fibroblast surface markers CD73 and CD90 in the isolated cells.m RNA transfection and puromycin selection results demonstrated the functional efficacy of the constructed m RNA screening system.Grayscale analysis revealed the extracellular cleavage efficiencies of the six sgRNA pairs as follows: 96.2 %,96.6 %,88.5 %,95.0 %,95.3 %,and 96.1 %.TIDE software analysis showed the following intracellular editing efficiencies for the sgRNAs: sgRNA1 3.3 %,sgRNA2 4.1 %,sgRNA3 10.6 %,sgRNA449.0 %,sgRNA5 5.8 %,and sgRNA6 5.1 %.Gel electrophoresis confirmed the presence of target sequence integration across homology arms in monoclonal cells,and sequencing results verified the successful creation of a positive heterozygous OCT4-EGFP-Puro R knock-in in Bama pig fetal fibroblasts.Conclusion:(1)Successful isolation of Bamah pig fetal fibroblasts from different genders was achieved.(2)The combination of the Puro R-m Cherry m RNA selection system and cell transfection strategies effectively enriched the population of transfected positive cells.(3)A robust technical platform for targeted gene knock-in was established using CRISPR/Cas9-mediated integration,with the insertion of EGFP-Puro R upstream of the stop codon in the fifth exon of endogenous OCT4.(4)Treatment with 5 μmol/L SCR7 during gene integration significantly enhanced the efficiency of homology-based gene knock-in in pig fetal fibroblasts. |
PDF Full Text Request