64 results about "Cloning embryos" patented technology

The invention provides an overexpression porcine co-stimulatory 4-1BB vector and application thereof. PCR (polymerase chain reaction) amplification is performed on a left homologous arm and a right homologous arm of an intron 1 of a rosa26 gene, a 4-1BB regulatory sequence and an OCT4 specific promoter; the left homologous arm, a 4-1BB expression cassette, LoxP locus-contained Cre and Neo expression cassettes, the right homologous arm and negative selection DTA diphtheria toxin are connected in sequence to obtain a 4-1BB homologous recombinant vector p4BOCNDR; the vector and a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated) targeting vector of sgRNA (small guide ribonucleic acid) containing the intron 1 of the specific targeting porcine rosa26 gene are transferred together into a porcine fetus fibroblast; by taking a positive cell as a donor cell and an oocyte as a recipient cell, a cloned embryo is obtained through a somatic cell nuclear transfer technique; the cloned embryo is transplanted into a porcine uterus for fetation to obtain a transgenic pig integrating a 4-1BB gene at the fixed point of a first intron of the rosa26 gene and automatically deleting a marker gene.

The invention provides a production method for Fbxo40 gene knockout pigs. The production method comprises the steps that a CRISPR-Cas9 targeting vector and a PGK-Neo resistant gene are jointly transfected into a porcine embryonic fibroblast to obtain a G418-resistant positive monoclonal cell, an Fbxo40 gene in the positive monoclonal cell generates insertion or deletion mutation, a reading frame generates frame shift and stops in advance, then the obtained cell is cloned to serve as a donor cell for nuclear transfer, and an oocyte serves as a receptor oocyte for nuclear transfer; cloned embryos are obtained through a somatic cell nuclear transfer technique, the high-quality cloned embryos are transferred into the fallopian tube of an oestrous sow, and the Fbxo40 gene knockout pigs are obtained through whole-period development. According to the production method, the Fbxo40 knockout pigs are efficiently obtained through a CRISPR-Cas9 gene editing technique at the low cost, and animal models are supplied to research of muscle development and diseases related to the muscles.

The invention discloses a method for breeding transgenic livestock rich in polyunsaturated fatty acid. The method comprises the following steps of: guiding DNA molecules which have a sequence 1 and a sequence 2 in a sequence list into somatic cells of a mammal to obtain transgenic cells in which a delta-12 fatty acid dehydrogenase and an omega-3 fatty acid dehydrogenase are expressed; cloning embryos by using the transgenic cells as nuclear transfer donor cells and using isolated ovocytes as nuclear transfer acceptor cells by nuclear transfer technology; and transplanting the cloned embryos into a uterus of the livestock by a non-operation method for gestation to obtain the transgenic livestock. The method of the invention can ensure that the delta-12 fatty acid dehydrogenase and the omega-3 fatty acid dehydrogenase are stably expressed in the body of a transgenic pig and that the content of omega-3 polyunsaturated fatty acids (PUFAs) in meat of the livestock is improved obviously.

The present invention discloses a cloned embryo treatment liquid, which comprises BIX-01294 and a PZM culture liquid, wherein the BIX-01294 concentration in the PZM culture liquid is 5-500 nM, and 500 mL of the PZM culture liquid comprise 3.156 of NaCl, 1.053 g of NaHCO3, 0.373 g of KCl, 0.024 g of KH2SO4, 0.049 g of MgSO4, 0.308 g of calcium lactate, 0.011 g of pyruvic acid, 0.073 g of L-glutamic acid, 0.273 g of hypotaurine, 10 mL of BME, 5 mL of MEM, 0.033 g of penicillin, 0.025 g of streptomycin, and the balance of Sigma water. According to the present invention, with the application of the cloned embryo treatment liquid using the technical scheme to perform the culture treatment on the cloned embryo, the gene expression of SUV39H1, SETdb1, DNMT1 or DNMT3a in the cell can be significantly reduced, and the H3K9me2 levels of 2cell and 4cell at the early embryo development stage can be significantly down-regulated.

The present invention provides a preparation method for an anti-porcine reproductive and respiratory syndrome cloned pig, comprising: transferring CRISPR / Cas9 targeting vectors and CD163 gene homologous recombination modification vectors into fibroblasts of a pig to obtain positive clone cells, a seventh exon of porcine endogenous CD163 gene being replaced with a eleventh exon of human CD163-L1 gene, so that the positive clone cells are incapable of mediating invasions of PRRSV; taking the positive cell as nuclear transfer donor cells and oocytes as nuclear transfer recipient cells to obtain a cloned embryo by adopting a somatic cell nuclear transfer technology; and impregenating a pig by transferring the cloned embryo into the uterus of the pig, to obtain a cloned pig.

The invention discloses a HBD3 mammary gland specific expression vector and a constructed recombinant cell. The vector comprises a HBD3 gene with a signal transduction peptide, and CSN5 and CSN3 are set upstream and downstream of the HBD3 gene respectively for serving as regulating and controlling elements. The host cell of the recombinant cell is a bovine fetal fibroblast cell, an exogenous expression vector pARNG-HBD3 is integrated to a pseudo attP locus under the action of phiC31 integrase, positive cloning is realized through medicament screening, and an antibiotic screening marker between two homodromous LoxP sequences on the vector is removed through Cre recombinase treatment. A HBD3-containing bovine fetal fibroblast cell from which an antibiotic screening marker is removed is taken as a nuclear donor cell of a transgenic cloning embryo through nucleus transplantation, gene cloning embryos are obtained through SCNT (Somatic Cell Nuclear Transfer), and a milk cow which is free from a HBD3 transgenic gene of the antibiotic screening marker can be produced by transferring these embryos into a receptor cow.

The invention discloses a method for efficiently preparing high-quality cloned embryos, belonging to the field of embryo transplanting methods. The method provided by the invention comprises the following steps of: agitating and screening to obtain sheep oocytes in the same cell period and transplanting a supplier cell into the sheep oocytes through utilizing microscopy injection; then treating by utilizing a fusion solution and putting the treated embryo into a mature solution to be cultured to form a fused embryo; and chemically activating and then starting and reconstructing embryo cleavage. According to the method provided by the invention, the sheep oocytes which are in the same cell period and have the uniform mature time through an agitating method are acquired, a new method for picking the good sheep oocytes is provided and the mature rate of the sheep oocytes is improved. According to the method provided by the invention, the operation efficiency of nuclear transfer is improved by utilizing a method of simultaneously denucleating through extrusion and carrying out nucleus injection by utilizing the same injection needle. The fusion efficiency is improved to more than 95% by using the fusion solution which is developed by the method provided by the invention, the successful rate of the cloned embryo is greatly improved and the subsequent embryo development is not influenced. The method provided by the invention has the advantages of reducing the loss of the embryos in the process of preparing the cloned embryo and improving the efficiency of preparing the sheep cloned embryos; and the method provided by the invention is a method for efficiently preparing the sheep cloned embryos with good quality and has a wide application prospect.

The invention provides a culture solution for improving the development quality of cell cloning embryos and in-vitro fertilized embryos of a cow body and a culture method. The culture solution comprises the following components: BME (Basal Eagle Medium) with the volume fraction being 2%, MEM (Minimum Essential Medium) with the volume fraction being 1%, 1mmol / L of glutamine, 8mg / mL of BSA (Bovine Serum Albumin), 50mu g / mL of gentamicin and 50mu g / mL of cephalosporin. After the cow embryo is cultured to the 96th hour, KSR (Knockout Serum Replacement) with the volume fraction being 5% is added. The culture solution and the culture method provided by the invention have the beneficial effects that the culture solution is utilized for in-vitro culture of the cell cloning embryos and the in-vitrofertilized embryos of the cow body, a result shows that the development rate and the quality of the blastosphere are respectively higher than those of a control group; by application of the culture solution and the culture method, the cell cloning embryos or the in-vitro fertilized embryos of the cow body can be obtained with high efficiency.

The invention relates to the field of animal cloning methods, in particular to a method for improving the development efficiency of a porcine cloned embryo. The method mainly comprises the step of adding a uterine fluid of a sow into a basic culture solution for the porcine cloned embryo. By the method, the blastocyst rate during early development of the cloned embryo and the total number of cellsin a blastocyst are significantly increased; a transcriptome sequencing result of the early embryo shows that culture through addition of the uterine fluid of the sow reduces the number of differentially expressed genes between the cloned embryo and a normal in-vivo fertilized embryo; most importantly, through the addition of the uterine fluid of the sow, also increased the fertility rate of therecipient sow of the cloned embryos and the number of litters per litter, the parturition rate of a receptor sow with the cloned embryo and the number of live births are also increased, which means that by the method, the final efficiency of the in-vivo development of the cloned embryo is effectively improved.

The invention provides a solution and method for culturing bovine somatic cell cloned embryos. The culture solution comprises merchant oviduct culture solution for bovine somatic cell cloned embryos (mSOF), and comprises activin A as well, wherein the concentration of the activin A is 20 to 80 ng / mL. The culture method comprises: culturing a bovine somatic-cell granular cell monolayer under conventional conditions for 3 days; adding the activin A in a concentration of 20 to 80 ng / mL to the merchant oviduct culture solution for bovine somatic cell cloned embryos (mSOF) from the fourth day; obtaining the solution for culturing bovine somatic cell cloned embryos; and using the solution for culturing bovine somatic cell cloned embryos for 5 days to obtain the bovine somatic cell cloned embryos. The solution and the method have the advantages of improving the 8 / 16 cell rate, blastula rate and hatchability of embryos and significantly raising the gene expression level of Na / K-ATPase, Glut-1, ActR II, Smad2 and the like, and are particularly suitable for application in the field of bovine somatic cell cloning.

The invention discloses a somatic cell cloning method for mammals such as cows, dogs or sheep and culture fluid utilized in the method. The method comprises following mammal somatic cell cloning embryo processing steps: (1) injecting to-be-transplanted mammal somatic cells into denucleated oocytes, carrying out electro-fusion, then selecting successfully fused mammal somatic cell cloning embryos in 1 hour after electro-fusion, and carrying out activation; and (2) after the activation, transferring the mammal somatic cell cloning embryos into G1.3 culture fluid, and carrying out culturing. The invention further relates to the culture fluid used in the method. The method disclosed by the invention presents the excellent technical effects described in the description.

The invention relates to the technical field of animal transgenosis and xenotransplantation, in particular to a construction method of a CD55 transgenic large animal. The invention provides sgRNA for site-specific integration of an hCD55 gene at a safe harbor Rosa26 site of a large animal genome, provides an integration plasmid comprising the sgRNA, constructs a transgenic cell for site-specific integration of a human complement regulatory protein CD55 at the safe harbor Rosa26 site through a site-specific integration technology, further obtains a cloned embryo, and obtains a transgenic hCD55 gene pig after pregnancy. The transgenic pig obtained by the method disclosed by the invention is good in healthy state and can survive to be adult. The expression quantity of the hCD55 is high, the hCD55 can be used for xenotransplantation, stable inheritance and normal breeding can be realized, and verification is carried out in the F1 generation.

The invention discloses an additive for improving the in-vitro maturation quality of oocytes. The in-vitro culture quality of the oocytes is improved by adding a PF4 protein additive to an in-vitro oocyte culture solution. By improving the in-vitro culture quality of the oocytes, the additive can be applied to the fields of in-vitro maturation of the oocytes, somatic cell cloning technology and parthenogenetic embryos. On one hand, in-vitro maturation of the oocytes of various species can be promoted, in-vitro fertilized embryos and cloned embryos with better quality can be prepared, and in-vitro maturation culture of the oocytes of human assisted reproduction is facilitated. On the other hand, the prepared high-quality embryos and animals produced by the embryos are used for research in the fields of medicine, life science, animal husbandry and the like, and development of production practice is further promoted.

A nuclear transfer method is provided wherein nuclear DNA in whole or part is injected into enucleated oocytes. The method is suitable for different donor cells, and preferably ES cells.

The invention discloses a method for improving in-vitro development efficiency of porcine cloned embryos. The method comprises the following steps of: performing molecular identification on bone marrow-derived mesenchymal stem cells, and establishing a method capable of identifying the purity of the cells by using a few cells through a 6-channel flow type technology; performing gene modification on the porcine bone marrow mesenchymal stem cells to obtain porcine bone marrow mesenchymal stem cells for expressing multiple reprogramming factors; and separating out positive porcine bone marrow mesenchymal stem cells by using a fluorescent flow type separation method, continuously culturing the positive porcine bone marrow mesenchymal stem cells for 3 to 7 days, performing nuclear transfer on the cultured cells serving as nuclear donor cells, and studying the influence of 4RFs-3days, 6RFs-3days, 4RFs-7days and 6RFs-7days pMSC serving as nuclear donor cells on the improvement of the development efficiency of the porcine cloned embryos. The results show that the 4RFs-7days pMSC can well promote fission of the cloned embryos and formation of blastospheres, the cloned embryos can form a homogeneous state with uniform cell quantity, and a foundation is laid for efficiently cloning adult high-quality pig varieties in large scale.

The invention belongs to the field of animal somatic cell cloning methods, and in particular relates to a method for improving development efficiency of pig cloned embryos. A main idea of the method provided by the invention is to jointly regulate histone ubiquitination of the cloned early embryos, and modification degrees of histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 4 trimethylation (H3K4me3) to improve the development efficiency of the pig cloned embryos, wherein the combined regulation refers to simultaneous reduction of the histone ubiquitination and the modification degrees of the H3K9me3 and the H3K4me3. The method provided by the invention comprises the following main operation steps: obtaining the pig somatic cell cloned embryos; obtaining exogenous mRNA; and introducing the exogenous mRNA into the cloned embryos. The method provided by the invention simultaneously overexpresses USP29, KDM4A and KDM5B genes, significantly improves the blastocyst rate ofdevelopment of the pig cloned embryos and the total number of cells in blastocyst, and improves the birth efficiency of cloned pigs by 3.82 times.

The invention discloses a clone embryos transplanting method. In the method, a surrogate motherhood which is the same with the cytoplasm subspecies of the receptor of the clone embryos is selected for carrying out clone embryos transplanting. The method increases the compatibility between the clone embryos and the surrogate motherhood as well as can remarkably improve the conception rate of the clone embryos after being transplanted.

The invention relates to a new function of a porcine BTG2 (B-cell translocation gene-2) in anti-PRRS (porcine reproductive and respiratory syndrome) virus, and in particular provides application of porcine BTG2 gene in anti-PRRS virus. The application is as follows: inhibiting replication of PRRS virus in cells by over-expression of BTG2 gene in cells. The invention also provides a method for screening an anti-PRRS virus pig by utilizing the expression quantity of BTG2 gene, and a method for preparing a cloned embryo of over-expressed BTG2.

The invention discloses a complete pig Xist (X inactive specific transcript) gene sequence and a method for improving the pig cloning efficiency. The pig Xist gene sequence is obtained through electronically cloning the pig Xist gene sequence by utilizing a biological informatics method and checking and analyzing the sequence. The method comprises the following steps of: designing and synthesizing anti-Xist siRNA (small interfering ribose nucleic acid) sequences, screening the effective anti-Xist siRNA sequence by utilizing a molecular biological technology, utilizing anti-Xist siRNA to inhibit nuclear transfer reconstructed embryos or Xist gene expression of nuclear transfer somatic cell donors so as to improve the development quality of pig cloning embryos, and thus realizing a purpose of improving the pig cloning efficiency. According to the method, the complete pig Xist sequence and the anti-Xist siRNA sequence are obtained at first; and moreover, by utilizing the method, the ectogenic quality of the pig cloning embryos can be improved, and the improved pig cloning efficiency has a significant meaning in the fields of livestock production, scientific research or medical science.

The invention discloses a preparation and in-vitro culture method of interspecies-cloned yak embryos. According to the method, yak-cattle interspecies-cloned embryos are constructed by using yak mammary glandular cells as donor cells and cattle or milch cow enucleated oocytes as receptor cytoplasts by a somatic nucleus transplantation technique; and an electrical activation-chemical activation-reelectrical activation mode and a self-made sequential culture system are utilized to acquire the high-efficiency high-activity yak-cattle interspecies-cloned embryos. The method disclosed by the invention can increase the yak breeding speed, shorten the yak breeding cycle, lower the yak production cost, enhance the breeding efficiency, reduce the damage level of the grassland to some extent, and effectively protect the species resource of the yak. Besides, the invention has important meanings for promoting research of interspecies-cloned yak, transgenic cloned yak embryos and nucleo-cytoplasmic interaction.

The present invention discloses a method for improving porcine somatic cell nucleus transplantation embryo development efficiency, and provides applications of chaetocin, wherein the applications comprise at least one selected from (a1) preparation of a nucleus donor cell culture liquid for animal somatic cell nucleus transplantation, (a2) preparation of a reconstruction embryo culture liquid for animal somatic cell nucleus transplantation, (a3) culture of a nucleus donor cell for animal somatic cell nucleus transplantation, (a4) culture of a reconstruction embryo for animal somatic cell nucleus transplantation, and (a5) promotion of the formation of blastula from the reconstruction embryo in animal somatic cell nucleus transplantation. According to the present invention, the nucleus donor cell or reconstruction embryo in animal somatic cell nucleus transplantation is treated with the small molecule drug chaetocin, such that the occurrence of the somatic cell nucleus transplantation re-programming is promoted, the blastula development and the blastula cell number of the cloned embryo are significantly improved, and the whole efficiency of the porcine somatic cell nucleus transplantation technology is improved; and the method provides important significance for the improvement of the whole efficiency of the porcine somatic cell nucleus transplantation technology.

The invention provides a bovine cloned embryo freezing solution, a bovine cloned embryo thawing solution, a kit and bovine cloned embryo freezing and thawing methods, and belongs to the in-vitro embryo freezing technology. The freezing method comprises the following steps: putting a bovine cloned embryo into a basic solution, and balancing the embryo at a temperature of 24-25 DEG C for 5-10 minutes; putting the processed embryo into a first vitrification freezing solution to balance the embryo at a temperature of 24-25 DEG C for 4-6 minutes, putting the processed embryo into a second vitrification freezing solution to balance the embryo at a temperature of 24-25 DEG C for 4-6 minutes and putting the processed embryo into a third vitrification freezing solution to balance the embryo at a temperature of 24-25 DEG C for 35-45 seconds, and finally putting the processed embryo into liquid nitrogen to freeze. And the frozen bovine cloned embryo is put in a first thawing solution, the embryois thawed at a temperature of 37-39 DEG C for 50-70s, the thawed embryo is put in a second thawing solution, the embryo is balanced at a temperature of 24-25 DEG C for 4-6 min, and finally placing theprocessed embryo is put in a base solution, and the embryo is balanced at a temperature of 24-25 DEG C for 4-6 min. With the method disclosed by the invention, the conception rate of the bovine cloned embryo is as high as 25%.

The invention discloses a preparation method of an anti-African swine fever cloned pig and belongs to the field of animal genetic engineering and genetic modification. A CRISPR / Cas9 gene knock-in technology is used, a CRISPR / Cas9 targeting vector and a pig Rosa26 gene donor vector are jointly transferred into a pig fibroblast to obtain a positive cell clone, a first intron of the pig Rosa26 gene in the positive clone is knocked in a gene capable of expressing sgRNA-Cas9 of a specific targeted knockout virus pE248R gene, such that the pE248R gene is knocked out in a targeted manner when viruses are propagated in vivo and a pig has ASFV resistance; a cloned embryo is obtained through a somatic cell nuclear transfer technology; and the cloned embryo are transferred into a pig uterus for pregnancy to obtain a cloned pig. The sgRNA-Cas9 system is knocked into a pig body for the first time and stably expressed to obtain ASFV resistance, such that a foundation is laid for breeding research on African swine fever resistance.

This invention is the first cat cloned by nuclear transfer, as well as any subsequent progeny produced by regular sexual reproduction. Nuclear transfer methods and techniques involving feline somatic cells, nuclei, or nuclear DNA, and the products thereof, are disclosed. In a single experiment 3 cloned embryos derived from the cumulus cell line and 2 embryos derived from fibroblast cells were transferred into a recipient female cat. Following embryo transfer after the period of gestation, a kitten was delivered. The cloned kitten has the exact DNA fingerprint as the cumulus cell line of the adult female cat from which the cumulus cell line was derived.

The invention discloses a method for treating a cattle somatic cell cloning embryo. Specifically, the method comprises the following steps: 1) injecting a to-be-transplanted cattle somatic cell into a de-nucleated oocyte and electrically fusing, selecting successfully fused cattle somatic cell cloning embryos after electrically fusing for 1 h and then performing activation treatment; and 2) after performing activation treatment on the cattle somatic cell cloning embryos, transferring into a G1.3 culture solution and culturing. The invention also relates to a culture solution used in the activation process of the cattle somatic cell cloning embryo and the culture solution used in an in vitro culture process of the cattle somatic cell cloning embryo. The method disclosed by the invention shows excellent technical effects as described in the description of the invention.

The invention discloses a method for in-vitro cloned embryo transplantation of a pregnant sow. The method comprises the following steps: feeding an estrus synchronization agent to each sow at a fixedpoint every day, after continuously feeding each sow with the estrus synchronization agent for 18 days, performing intramuscular injection of PG600 5 days later, and selecting an individual with normal estrus for in-vitro cloned embryo transplantation 1 day later. According to the method, a proper microenvironment is provided for the pregnant sow after the cloned embryo is transplanted by virtue of feeding or injection of the estrus synchronization agent and the PG600, synchronism of the estrus, ovulation and embryo transplantation time of the pregnant sow is controlled, and the success rate of cloned embryo implantation and the pregnancy rate of the pregnant sow are greatly increased, so that batch production of cloned pigs is realized, and a foundation is laid for large-scale productionof the cloned pigs.

The invention belongs to the technical field of biological engineering and discloses a preparation method of pig cloning embryos. The preparation method comprises the following steps of: obtaining donor cells; taking recombinant vectors of reprogramming factors, transferring into the donor cells and obtaining nuclear donor cells; transplanting the nuclear donor cells into egg cells with removed cell nucleuses, and carrying out fusion and activation to obtain the pig cloning embryos. The preparation method provided by the invention has the advantages that the nuclear donor cells are directly transplanted into egg cells with removed cell nucleuses after being obtained, the step of utilizing the reprogramming factors to prepare the pig cloning embryos is reduced, the operation is simple and easy, the working period is shortened and the growing efficiency of the pig cloning embryos is effectively improved.

The invention discloses a method for improving bovine somatic cell cloning efficiency. By inducing the expression of zfp57 gene, the methylation of imprinted genes in the development process of a bovine cloned embryo is restored to a normal level, so that the cloning efficiency of bovine somatic cells is improved. The method for improving the bovine somatic cell cloning efficiency provided by theinvention utilizes the zfp57 to correct the abnormal low methylation of imprinted genes on the bovine cloned embryo, and makes the methylation level close to that of in vitro fertilization embryos soas to improve the embryo quality of the bovine cloned embryos, and to promote the development of the bovine cloned embryos. The method provides a theoretical basis for clarifying the mechanism of abnormal methylation imprinting in the process of reprogramming somatic cell-cloned embryos, and the reason of low efficiency of somatic cell cloning.