CRISPR/Cas9-mediated specific integration of fat-1 at the goat MSTN locus.

Recent advances in understanding the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, no study has reported simultaneous knockout of endogenous genes and site-specific knockin of exogenous genes in large animal models. Using the CRISPR/Cas9 system, this study specifically inserted the fat-1 gene into the goat MSTN locus, thereby achieving simultaneous fat-1 insertion and MSTN mutation. We introduced the Cas9, MSTN knockout small guide RNA and fat-1 knockin vectors into goat fetal fibroblasts by electroporation, and obtained a total of 156 positive clonal cell lines. PCR and sequencing were performed for identification. Of the 156 clonal strains, 40 (25.6%) had simultaneous MSTN knockout and fat-1 insertion at the MSTN locus without drug selection, and 55 (35.25%) and 101 (67.3%) had MSTN mutations and fat-1 insertions, respectively. We generated a site-specific knockin Arbas cashmere goat model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer for the first time. For biosafety, we mainly focused on unmarked and non-resistant gene screening, and point-specific gene editing. The results showed that simultaneous editing of the two genes (simultaneous knockout and knockin) was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in safe animal breeding.

Identification and characterization of an endoplasmic reticulum localization motif.

Sorting motifs are involved in the transport of diverse proteins. In the present study, we identified a hydrophobic peptide (WRPWRNFWWSIRVPWRRN) that was able to target enhanced green fluorescent protein- or DsRed2-enriched vesicular-like sub-compartments of the endoplasmic reticulum (ER). Analysis of mutation constructs revealed that the sequence WRPWRNFWW was responsible for the ER-targeting activity, and the arginine residue of the peptide is a critical determinant of ER localization. Results from co-immunoprecipitation, glutathione S-transferase pull-down, liquid chromatography-tandem mass spectrometry, and western blotting analyses demonstrated that this motif could bind with the γ2-COP subcomplex of coat protein complex I (COPI), which is involved in the retrieval and transport of ER-resident proteins from the Golgi apparatus to the ER. Overall, we report a new hydrophobic peptide that possesses an arginine-based ER localization motif, which can help elucidate the mechanisms of ER sorting mediated by COPI.

Enhanced expression of MYF5 and MYOD1 in fibroblast cells via the forced expression of bos taurus MYF5.

The formation of vertebrate skeletal muscles widely thought to be under the control of hierarchy of regulatory genes. MYF5 is one of the myogenic determination gene expressed in the developing mouse dermomyotome which control skeletal muscle differentiation. In the current work, we had obtained the cDNA sequence including the full coding region of the bos taurus myogenic factor MYF5 by reverse transcription polymerase chain reaction. Furthermore, we examined whether fibroblast cell derived from mouse and bos taurus can be transduced using plasmid vectors carrying bos taurus MYF5. Bos taurus MYF5 activates MYF5 and MYOD1 expression after 1 day culture. The concerted upregulation of the myogenic regulatory factors enhanced myosin (skeletal fast) expression. These observation show that MYF5 is essential for myogenic differentiation and provides candidates for regulation bos taurus skeletal muscle development.