ABSTRACT
This study aims to knock out the goat β-lactoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus, and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency. First, we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG. This sgRNA vector was then transfected into goat ear fibroblasts (GEFs), and the target region was examined by T7EN1 assay and sequencing. Second, we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus. This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs. Transfected cells were then treated with 0, 5, 10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency. Next, we used 800 μg/mL G418 to screen G418-resistent cell clones, and studied hLF site-specific knock-in cell clones by PCR and sequencing. The editing efficiency of sgBLG was between 25% and 31%. The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner, which could reach 3.5-fold compared to the control group. The percentage of positive cells with hLF knock-in was increased to 32.61% when 10 μmol/L RS-1 was used. However, when the concentration of RS-1 increased to 20 μmol/L, the percentage of positive cells decreased to 22.22% and resulted in an increase of senescent cell clone number. These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system, and optimum concentration of RS-1 could improve knock-in efficiency, which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of immunization with prokaryotically expressed recombinant fusion protein of extracellular near-transmembrane domain of Tibet minipig leptin receptor (OBR) on fat deposition in SD rats.</p><p><b>METHODS</b>A pair of specific primers containing BamHI and HindIII restriction enzyme sites was designed to amplify the extracellular near-transmembrane domain (1705-2364 bp) of Tibet minipig OBR gene. After digestion, the amplified fragment was inserted into the plasmid pRSETA between BamHI and HindIII sites. The recombinant plasmid was transformed and expressed in E.coli BL21(DE3) and the product was analyzed by SDS-PAGE and Western blotting. SD rats were immunized with the fusion protein, and the changes in body weight, feed intake, body length, Lee's index, percentage of abdominal fat, liver fat deposition and subcutaneous fat deposition were assessed.</p><p><b>RESULTS</b>The recombinant fusion protein obtained (about 27.6 kD) was expressed in E.coli induced by IPTG and identified by SDS-PAGE and Western blotting. The rats immunized with the fusion protein showed no significant changes in body weight, body length, Lee's index, percentage of abdominal fat or liver fat deposition as compared with the control rats. Nevertheless, the immunization caused significantly increased feed intake and significantly decreased volume of subcutaneous fat cells.</p><p><b>CONCLUSION</b>Immunization with the fusion protein of extracellular near-transmembrane domain of Tibet minipig OBR can promote feed intake and suppress subcutaneous fat deposition in SD rats.</p>