6 results about "Nuclear transfer" patented technology

The invention discloses a culture solution for improving the development rate of in-vitro cultured pig blastocysts as well as application and a use method of the culture solution, and belongs to the technical field of livestock embryo engineering. The invention discloses a culture solution for improving the development rate of in-vitro cultured pig blastocysts, which is characterized in that a PZM3 culture solution is used as a basic culture solution, and 5-25ng / mL of tumor necrosis factor superfamily cell factor TNFSF12 is added. According to the present invention, the pig embryo needs to be subjected to in-vitro culture for 4 days in the TNFSF12-free basic culture solution, and then is transferred to the TNFSF12-containing basic culture solution so as to be continuously cultured for 2 days, such that the generation efficiency of the pig blastocyst can be effectively improved, and the support is provided for the application of the pig in-vitro fertilization, the somatic cell nuclear transfer, the embryo genome selection and other embryo engineering technologies in the agricultural breeding and the human medicine;

The invention discloses a method for improving pig somatic cell nucleus embryo transfer efficiency, and belongs to the technical field of pig somatic cell nucleus transfer. The method comprises the following steps: treating a reconstructed embryo subjected to pig somatic cell nuclear transfer by using sodium butyrate to improve the acetylation level of the reconstructed embryo, or overexpressing KDM2A to reduce the methylation level of the reconstructed embryo so as to improve the efficiency of transferring the embryo by using the pig somatic cell nuclear transfer. According to the present invention, the research results show that the high enrichment of the ZGA gene promoter region H3K36me2 and the enrichment reduction of the far-end cis-regulatory element H3K27ac result in the existing unrepresented epigenetic disorder, such that the SCNT efficiency is limited; therefore, the H3K36me2 is deleted through the demethylase KDM2A, or the HDAC for inhibiting the deacetylation of the H3K27ac in a NaB-mediated manner is used, so that the development arrest of the SCNT embryo is relieved, and the blastocyst forming efficiency is improved.

The application discloses application of sodium crotonate in improving bovine embryo development potential. In the process of culturing bovine SCNT embryos in vitro, adding sodium crotonate (Nacr) with a proper concentration can promote the development of bovine SCNT cells, significantly improve the development rate and blastocyst rate of the SCNT embryos, improve the embryo quality, and further improve the somatic cell nuclear transfer efficiency of pre-implantation embryos. On this basis, the inventors construct a bovine SCNT early embryo in-vitro culture system, optimize the formula of the in-vitro embryo culture solution and the concentration of Nacr, and further establish a method for improving the development potential of early bovine embryos in vitro. The application can improve the development quality of bovine SCNT embryos, provides a research basis for the rapid propagation of high-yield dairy cattle, high-quality beef cattle and even disease-resistant fine breeds, provides a reference for the batch production of high-quality bovine embryos in vitro, and has important practical significance in the field of SCNT embryo in-vitro culture.

The invention relates to the technical field of animal gene engineering and biology, in particular to a pig MSTN, pAPN and CD163 gene synchronous editing embryo and a preparation method and application thereof. On the basis of a CRISPR / Cas9 technology, an MSTN exon 1, a CD163 exon 7 and a pAPN exon 2 are selected as targeting areas, efficient sgRNA is designed and screened, an integrated tandem editing vector containing a three-gene sgRNA expression cassette is constructed through homologous recombination, after pig embryo fibroblasts are electrically transfected, puromycin screening and sequencing identification are performed to obtain positive PEF cells, and the positive PEF cells are subjected to high-throughput screening and high-throughput screening. And performing somatic cell nuclear transfer to prepare and synchronously edit embryos. The synchronous editing efficiency of the three genes reaches 8.9%, the fusion rate of the edited embryos is larger than or equal to 70%, the cleavage rate is larger than or equal to 75%, the blastocyst rate is larger than or equal to 12%, the developmental ability is normal, synchronous improvement of genes related to growth performance and disease resistance is achieved, and the method has important industrial application value.

The invention discloses an application of lactobacillus johnsonii in promoting in-vitro maturation of porcine oocytes, and a synergistic regulation system is constructed by optimizing formulas of an oocyte maturation culture solution, an electric activation solution and an embryo culture solution. The application can effectively promote the in-vitro maturation rate of the porcine oocytes to be increased from 68.84% to 77.60%, promote the discharge of first polar bodies, reduce the apoptosis rate of the oocytes and improve the development potential of the oocytes after nuclear transplantation. Therefore, the in-vitro maturation efficiency of the oocytes is improved, the problems of low maturation efficiency, high apoptosis ratio and long culture period at present are solved, and the technical system can provide more high-quality oocytes for somatic cell nuclear transplantation, manual cloning, in-vitro fertilization, transgenic cloning, gene editing and the like, and has a wide application prospect in actual production.

The invention relates to a system for MSTN / IGF2 double-gene editing and a preparation method and application thereof. By designing and screening an efficient spacer sequence aiming at the MSTN and IGF2 genes and utilizing a Cas12f system to carry out double-gene editing, the MSTN and IGF2 double-gene edited cell line is successfully obtained. Further preparing a cloned embryo through a somatic cell nuclear transfer technology, and transferring the cloned embryo to finally obtain the double-gene edited calf with remarkable phenotype. Experimental results show that the birth weight of the edited calves reaches 55 kg and is remarkably higher than that of the wild calves (31 kg), it is proved that the double-gene editing strategy has the remarkable effect on promoting the growth and weight of the beef cattle, and a new genetic material and a new technical path are provided for beef cattle breeding.