Pegm t easy vector

To measure the efficiency of CCR5 genomic mutation, we performed T7 endonuclease 1 (T7E1) assay [79 (link)] and sequencing analysis as previously described [66 (link)]. Briefly, according to the protocol of Blood & Cell Culture DNA Midi kit (Tiangen, China), genomic DNA was extracted from the modified cells and PCR amplification of CCR5 gene with a set of primers (Additional file 6: Table S3). The PCR products were purified by Gel Extraction Kit (Promage). Then we used 300 ng purified PCR products combined with 2 µl 10 × NEB buffer (New England BioLabs) and appropriate deionized H₂O to make the final volume of 20 µl. The mixture was annealed to form the heteroduplexes and digested with five unites of mismatch-sensitive T7E1 (New England BioLabs) for 1 h at 37 °C [79 (link)]. The digested DNA was analyzed by 1.5% agarose gel and the editing frequency was quantified by Image J software as described previously [17 (link)]. The PCR products were also inserted into pEGM-T Easy Vector (Promega) for sequencing by a T7 primer.

The sheep LHβ cDNA sequence (GenBank Accession no. NM_001009380.1) was employed as a template. Primer pairs were designed based on the whole genome and coding sequence region of the LHβ gene (Table 1). The functional domains of the deduced proteins were analyzed with the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) [16 (link)]. The homologies of LHβ coding products in different species were analyzed by ClustalW2 online (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
The RNA used in this process was extracted from the tissues of an adult female Chinese indigenous Small-Tailed Han Sheep (Gansu Province, China) with TransZol (TransGen Biotech, Beijing, China). RT-PCR (reverse transcription PCR) was performed by using Taq polymerase (TransGen Biotech, Beijing, China) and TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Following separation by agarose gel electrophoresis, the predominant PCR product was purified and subsequently cloned into the pEGM-T-Easy vector (Promega, WI, USA) prior to sequencing.