134 results about "Somatic cell nuclear transfer" patented technology

The present invention provides cloned, genetically modified, endothelial cells, and the stem cells from which they are derived, which are produced by somatic cell nuclear transfer. The invention further provide novel therapeutic methods in which such cells are administered to a patient with tumors to inhibit and / or disrupt angiogenesis of the tumors, thereby inhibiting tumor growth and killing tumor cells.

The invention relates to the field of genetic engineering and genetic modification, in particular to a method for obtaining an MC4R gene knockout pig by using a CRISPR / Cas9 system for editing MC4R genes through a somatic cell nuclear transfer technology. sgRNA is designed by aiming at two sites (a 6133-6152bp sequence and a 7127-7146 bp sequence in a CDS region of the MC4R gene) in the CDS region of the pig MC4R gene for the first time; in addition, the CRISPR / Cas9 system is used for simultaneously cutting the two sites; the large segment deletion of the MC4R gene is realized; in addition, the gene knockout individual pig subjected to large segment deletion is obtained; a feasible method is provided for studying the pig MC4R gene.

The invention provides application of a Trim63 gene in preparation of a high muscle content and hypertrophic cardiomyopathy model cloned pig. Somatic cells of the Trim63 gene of a modified pig are utilized as nuclear transfer donor cells, oocytes are utilized as nuclear transfer recipient cells, and a cloned embryo is obtained through the somatic cell nuclear transfer technology. The cloned embryo is transferred into a pig uterus to gestate to obtain the Trim63 gene modified high muscle content and hypertrophic cardiomyopathy model cloned pig, artificial intervention methods, such as operation and so on for the cloned pig are not needed any more, and the efficiency of establishment of a disease model is improved. According to the high muscle content and hypertrophic cardiomyopathy model cloned pig, paired Cas9n targeting vectors are utilized for the first time to perform gene editing for large animals. The method is low in cost, sharply shortens the time for obtaining a homozygote pig and lays a foundation for gene function research and the disease model establishment for the large animals by utilizing the CRISPR / Cas9 technology.

The present disclosure provides methods of producing enucleated cells by photoablation. Such enucleated cells may be used as recipient cells for Somatic Cell Nuclear Transfer and cloning. The nuclear donor and / or enucleated recipient cells may be any fish cells, such as zebrafish, koi, or medaka fish cells. Such methods may be used to efficiently produce transgenic fish including by way of example zebrafish, koi, and medaka fish.

Methods for making human ES cells and human differentiated cells and tissues for transplantation are described, whereby the cells and tissues are created following somatic cell nuclear transfer. The nuclear transfer donor is genetically modified prior to nuclear transfer such that cells of at least one developmental lineage are de-differentiated, i.e., unable to develop, thereby resolving the ethical dilemmas involved in reprogramming somatic cells back to the embryonic stage. The method concomitantly directs differentiation such that the desired cells and tissues may be more readily isolated.

The invention belongs to the technical field of pig genetic breeding, and specifically relates to a culture solution capable of improving in vitro maturation rate of pig oocytes and application thereof. The culture solution capable of improving the in vitro maturation rate of pig oocytes is prepared from the following raw materials with the following concentrations: 9.5 g / L of TCM-199, 2.2 mg / mL of NaHCO3, 3.05 mmol / L of D-glucose, 0.91 mmol / L of Sodium Pyruvate, 0.5 g / L of Streptomycin sulfate, 0.05% of PVA, 0.57 mmol / L of Cysteine and 0.075 g / L of Penicillin G. The 0.05% of PVA and 5% of a follicular fluid are added on the basis of pig oocyte in vitro maturation culture solution widely used in our country, so that the in vitro maturation rate of the pig oocytes can be improved to more than 90%, and matured oocytes with higher quality and more quantity can be provided for porcine somatic cell nuclear transfer technology.

The invention provides a bovine oocyte in vitro maturation culture solution and a culture method. The maturation culture solution consists of 10% of fetal calf serum and 1% of ITS-G, as well as 0.075IU / mL of HMG, 1[mu]g / mL of 17[beta]-estradiol, 10ng / mL of EGF, 2.2mg / mL of bFGF and 50ng / mL of CXCL12. The culture solution, when applied to bovine oocyte in vitro maturation culture, can improve maturation quality of bovine oocyte in vitro culture, a development rate of in vitro fertilized embryos as well as a development rate of somatic cell nuclear transplantation embryos and blastocyst quality.

The invention relates to a method and equipment for femtosecond laser somatic cell nuclear transplantation, in particular to a micro-nano control method which adopts a light knife effect induced by femtosecond laser two-photon and optical tweezers gradient force induced by high photon density. A pump laser is started-up; the laser produced by the pump laser is introduced into a resonator of a femtosecond laser; when the oscillation is done, the energy of the ultrashort femtosecond laser pulse is amplified through a regenerative amplifier, and the pulse number and the repeated frequency of laser output are chosen. The emitted femtosecond laser is divided into two beams of light, one of which used for realizing the femtosecond optical tweezers, and the other of which is used for realizing the function of the femtosecond optical tweezers through a half-permeable and semi-reflecting mirror, and then the two beams of light are coupled with an optical path through another half-permeable andsemi-reflecting mirror to realize the amplification of a spot light area; then the light beams is focused on a target cell, and the harmless transplantation operation of the somatic cell nuclear is done. A switch for controlling a light valve is arranged in a software control system, which can not only realize the operation of the laser spot on the target cell, but also adjust the scanning speed and change the exposure time to meet different requirements and enhance the success rate of the somatic cell nuclear transplantation.

The invention belongs to the field of genetic engineering, and provides a method for knocking out a selective marker gene of a transgenic pig; the method disclosed by the invention comprises the following steps: promoting a Cre / loxp site specific recombinase system by utilizing a pig sperm specific included promoter, establishing a self-shearing component PCN (Personal Communication Network), normally carrying out targeting and screening positive clone, after carrying out SCNT (Somatic Cell Nuclear Transfer) to obtain founder positive transgenic pig, and self-shearing to knock out the marker gene through hybridization, wherein in the self-shearing component PCN, P represents the pig sperm specific promoter; C represents a Cre enzyme gene; and N represents a screened marker neo gene required while targeting. Because Cre enzyme is specifically promoted after sperm is mature, the pig, the selective marker neo gene of which is knocked out, can be obtained; and thus, the pig, the selective marker gene of which is knocked out, can be conveniently obtained.

The invention discloses a preparation method of liver cells with low expression or no expression of PERV. The preparation method includes the following steps: (1), respectively preparing a recombinant vector containing siRNA applied to RNA interference technology, a recombinant vector containing gRNA applied to CRIPRS / Cas9 technology and a vector containing Cas9 applied to CRIPRS / Cas9 technology; (2), jointly transfecting the three vectors to porcine somatic cells, culturing the transfected porcine somatic cells, and screening and identifying to obtain monoclonal genetically-modified porcine somatic cells; (3), utilizing somatic cell nuclear transfer technology to obtain genetically-modified pig containing nuclear genes of the genetically-modified porcine somatic cells; (4), extracting liver cells of the genetically-modified pig. According to the preparation method, knockout before transcription and inhibition after transcription of PERV genes are realized, and the technical scheme is effective and feasible.

The invention relates to a coronavirus resistant clone pig based on CRISPR / Cas9 gene editing technology and a preparation method thereof. The preparation method of the clone pig is characterized by comprising the following steps of: (1) obtaining gRNA sequences of a second exon and third exon of a specific targeting pAPN gene, wherein the nucleotide sequencesare shown as SEQ ID No. 1-2 in sequence; preparing CRISPR-Cas9 targeting vectors PX330-pAPN2 and PX330-pAPN3 corresponding to the gRNA sequences; (2) transferring the obtained targeting vector PX330-pAPN2 or targeting vector PX330-pAPN3 into porcine fetal fibroblasts to obtain a pAPN gene editing cell line; (3) carrying out somatic cell nuclear transfer operation on the pAPN gene editing cell line, transferring the reconstructed embryointo a substitute pregnancy receptor, and bearing the clone pig through natural pregnancy. The method can obtain the clone pig with complete deletion expression of porcine coronavirus receptor pAPN,and has the capability of resisting porcine coronavirus infection.

A genetic engineering technology for preparing the clonal pig by gene knock-out and somatic cell nucleus transplantation includes such steps as homoglous recombining to destroy the muscle growth inhibin gene of somatic cell, externally screening the targeted cells, and nucleus transplanting. Its advantage is stable inheritance of excellent characteristics.

The invention discloses a method for producing recombinant human BChE (butyrylcholinesterase) from transgenic non-human mammals by using gene knock-in and nuclear transfer technologies, and particularly provides a donor construct used for specific expression of a mammary gland. The construct integrates DNA (deoxyribose nucleic acid) coding sequences of human BChE to a specific expression protein gene locus of the mammary gland in targeted manner and performs successful transfection on non-human mammal embryo fibroblasts, juvenile and adult somatic cells or embryonic stem cells, clone is performed by using a somatic cell nuclear transfer technology so as to obtain a new individual, and the carried human BChE gene enables the mammary gland of a non-human mammal to express stably and secrete holoenzyme or a monomer of the human BChE. Recombinant protein produced with the method can be used for preventing and treating nerve poison and organic phosphorus compound poisoning as well as apnea caused by cocaine poisoning and succinylcholine, and for detecting and removing residues of organic phosphorus compounds on vegetables, other crops, various article layers and the soil.

The invention provides a CD28 gene overexpression vector and application thereof. A target biological genome serves as a template, left and right homologous arms of a first intron of an ROSA26 gene, a CD28 gene expression frame and an OCT4 gene promoter are amplified through PCR (polymerase chain reaction), and the left homologous arm, the CD28 gene expression frame, LoxP-locus-containing Cre expression frame and Neo gene expression frame both regulated by the OCT4 gene promoter, the right homologous arm and a negative selection marker DTA are connected sequentially and are constructed to an eukaryotic expression vector to obtain the overexpression vector. The overexpression vector and a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) 9 target vector containing a first intron of a specific target pig ROSA26 gene are transplanted into a fibroblast of a pig fetus, a positive cell serves as a donor cell, an oocyte serves as a recipient cell, and a cloned embryo is obtained according to a somatic cell nuclear transfer technology; the cloned embryo is transplanted into the uterus of a pig for gestation to obtain a transgenic pig with a CD28 gene which is integrated at a fixed point in the first intron of the ROSA26 gene, wherein a marker gene of the transgenic pig is knocked out automatically.

The invention discloses a new method for gene injection for a somatic cell nuclear transfer reconstructed embryo. The method comprises the following steps: target gene preparation, somatic cell nuclear transfer and a microinjection technology. An expression vector is constructed after a target gene is obtained, the cell nucleus of an oocyte is removed by virtue of micromanipulation, then a somatic cell is injected in the space of the denucleated oocyte, then an exogenous gene is directly injected in the somatic cell, the reconstructed embryo is constructed through one-step electric fusion and activation, and the embryo is transferred to obtain a transgenic progeny. According to the method disclosed by the invention, large-fragment cell gene transfection and screening are avoided, the transgenosis efficiency is increased, the time is saved, and the transgenosis application range is expanded. Importantly, the somatic cell is injected in a transpanent zone during a nuclear transfer operation process, thus microinjection for the exogenous target gene is facilitated.

The invention provides a roboticized somatic cell nuclear transfer operation method based on a micro-channel. The method is characterized in that the introduced micro-channel is used for storing cells; parallel double needles are introduced and used for cell denucleation and nuclear injection respectively. The method comprises the following key steps of micro-channel storage of the cells, cell positioning, cell holding, cell denucleation, switching between the denucleation needle and the nuclear injection needle, nuclear injection of the cells and release of the operated cells. The experimental results show that compared with a traditional somatic cell nuclear transfer process, time is saved by 41.7% in the process, the surviving rate and the success rate are equivalent to those of manualsomatic cell nuclear transfer, and the somatic cell nuclear transfer efficiency and repeatability are effectively improved.

The present invention relates generally to the field of somatic cell nuclear transfer (SCNT) and to the creation of cloned animals and cells. The disclosure relates to a method of cloning a mammal, obtaining pluripotent cells such as embryonic stem cells, or for reprogramming a mammalian cell using an oocyte and a fertilized embryo.

The present invention provides for the production of homozygous primary cells that carry a specific transgenic integration of interest on both chromosomes by bypassing breeding. These cell lines can they be used for the accelerated production of homozygous transgenic animals by somatic cell nuclear transfer. The invention is thus useful in the production of transgenic ungulate animals capable of producing desired biopharmaceuticals in their milk at higher yield than a comparable heterzygote. By combining the selection techniques of the current invention with somatic cell nuclear transfer it can be applied to large animals, where there is a strong need to shorten the time to homozygosity.

The invention belongs to the mammal reproduction technology field, particularly relates to a method for obtaining cloned embryos with cow somatic cells and discloses a method for producing cow cloned embryos simply and efficiently. According to the research results that pig somatic cells dissolves and disappears automatically in thirty minutes after being injected into ooplasm, whole cells with sound membrane are extracted from cows as donors and are injected into the whole cell cytoplast directly to construct cow cloned embryos, simplifying the operational process of somatic cell nuclear transfer. The method is characterized in that: (1) selecting and cultivating cow ovarian cumulus- oocytes complex; (2) enucleating of the occytes; (3) preparing donor cells; (4) preparing co-cultivation cell monolayer; (5) constructing reconstructed fetus; (6) activating the reconstructed fetus; and (7) cultivating the reconstructed fetus. The method has the best feather of easy operation, simple steps, stable results, providing a new idea and method for the expansion and the further study of cow somatic cells nuclear transplanting technology.

The invention belongs to the technical field of animal genetic engineering and particularly relates to a method for improving pig meat quality. The method is characterized in that a peroxisome proliferator-activated receptor (PPAR) gamma gene serves as an important candidate gene for improving the meat quality, a fibroblast cell line of a 30-day-old fetus of a large white pig is established, a SwaI linearized pN1-MCK-PPAR gamma2 expression vector is shifted to the 30-day-old fetus fibroblast cell line of the large white pig through an electrotransfection method, and a PPAR gamma transgenic pig is prepared in a method of somatic nucleus transplantation. The influence of muscle tissue overexpression PPAR gamma genes on meat traits such as intramuscular fat deposition is verified in a transgenic pig, the contradiction of simultaneously selecting meat quality and meat quantity in conventional animal breeding is overcome, and the novel method is provided for cultivating lean meat pigs with good meat quality.

The present invention relates generally to the field of somatic cell nuclear transfer (SCNT) and to the creation of cloned animals and cells. The disclosure relates to a method of cloning a mammal, obtaining pluripotent cells such as embryonic stem cells, or for reprogramming a mammalian cell using an oocyte and a fertilized embryo.

The invention discloses an expression vector for a piggyBac transposon and a transgenic pig and a construction method thereof, belonging to the field of gene engineering. The expression vector is constructed by connecting a fusion fragment containing a loxP-Neo / EGFP gene expression cassette to pCyL50 plasmid. The vector is introduced into the genome of a pig, and somatic cell nuclear transfer technology is employed to produce the transgenic pig. The expression vector provided by the invention has the piggyback transposon, greatly improves transformation efficiency, changes a common plasmid series recombination transgene integration model into a single-site single-copy integration model and better simulates the internal environment of a biological gene. The construction method for the transgenic pig provides a novel approach for preparation of a variety of transgenic animals and a simple, convenient and highly efficient approach for fabrication of biological models of transgenic animals.

The invention discloses an expression vector resisting PCV2 and a transgenic pig and construction methods thereof, belonging to the field of gene engineering. The expression vector is constructed by respectively connecting an H1 promoter and a pPB-sh2 sequence to a pUC57-H1 plasmid and then connecting a fusion fragment containing a loxP-Neo / EGFP gene expression cassette to pPB-H1-sh2. The vector is introduced into the genome of a pig, and somatic cell nuclear transfer technology is employed to produce the transgenic pig. The expression vector provided by the invention has a piggyback transposon, greatly improves transformation efficiency, changes a common plasmid series recombination transgene integration model into a single-site single-copy integration model and better simulates the internal environment of a biological gene. The constructed transgenic pig degrades PCV2mRNA through siRNA synthesized by itself, thereby fundamentally inhibiting PCV2 infection.

The invention provides a method for knocking out a cattle beta-lactoglobulin gene by using zinc finger nucleases (ZFNs), comprising the following steps of: according to a cattle beta-lactoglobulin gene sequence, designing ZFNs specific site expression vector and transplanting the ZFNs specific site expression vector into cattle fibroblast; and obtaining cells with the beta-lactoglobulin gene knocked out. By using ZFNs mediated gene knockout, one-time transfection can be realized so as to obtain cell clones with biallelic genes knocked out, which is difficultly achieved in the conventional gene targeting process. The drug screening process is saved. The method disclosed by the invention is advantageous for forming monoclonal cells. The process required by cells for resisting drug toxic process is avoided. The method plays a key role in the improvement of subsequent somatic cell nuclear transplantation efficiency and embryonic development quality. Simultaneously, resistance genes are not contained; and the biological safety evaluation process is greatly simplified.

The invention provides a method for separating cells from blood and cultivating the cells and a method for cloning non-human animals. The method for separating cells from blood and cultivating the cells comprises following steps: (1) adding the blood to an upper layer of a separation liquid and performing centrifugation in a manner of density gradient centrifugation for obtaining mononuclear cells; (2) inoculating the mononuclear cells onto an in-vitro cultivation device containing an adherent cell culture medium to obtain adherent cells; and (3) performing serial subcultivation to the adherent cells to obtain somatic cell nuclear transplanted donor cells. The somatic cell nuclear transplanted donor cells obtained through the method can be directly used for manually cloning the non-human animals. The method is simple in operation processes, is easy to carry out, is low in cost, is less in stress stimulation and harm in animals, and can ensure a complete appearance of the animals and improve animal welfare.

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 an optimized mice somatic cell nuclear transplantation method. According to the mice somatic cell nuclear transplantation method, three kinds of cells, including cumulus cell, fetal fibroblasts, and embryonic stem cell, are adopted to construct reconstructed embryos; the activation efficiencies of three reconstructed embryos in different activation mediums are compared, development efficiencies in different solutions are compared, and pregnancy efficiencies of different embryo transplantation methods are compared; it is concluded that calcium-free KSOM activation medium is a universal high efficiency activation medium, and the activation efficiency is about 93.5%; KSOM-AA culture medium is suitable for in vitro culture of the reconstructed embryos of cumulus cells and embryonic stem cells, and the best culture medium for fetal fibroblasts is alpha MEM culture medium; when the reconstructed embryos are at 2-cell period, nuclear transplanted animals can be obtained via transplantation of embryos through bilateral fallopian tubes, wherein for each fallopian tube, transplantation of 15 embryos is carried out. The optimized mice somatic cell nuclear transplantation method is capable of increasing reconstructed embryo cleavage rate, blastula development rate, cloning animal birth rate, and survival rate, so that the success rate of obtaining embryonic stem cells from the reconstructed embryos is increased.

The invention belongs to the technical field of animal gene engineering, and particularly relates to a marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and an application thereof. The marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector disclosed by the invention is relevant to the field of transgenic animals. A donor plasmid pcCAG-R26-PBD2-T2A-PBD2which can express dual-copy pbd-2 genes is constructed, and the donor plasmid further contains a neomycin resistant gene neoR expression box having loxP sequences on two sides. Experiment certificatesthat when the plasmid and a targeting plasmid pX330-pRosa26 which can express Cas9 protein and guide RNA targeting a porcine Rosa26 site at the same time are electrically shifted into recipient cellsat the same time, fixedpoint integration of a pbd-2 gene into the porcine Rosa26 site is realized; through Cre recombinase, resistant marking is deleted; and through somatic nucleus transplantation and embryo transplantation, pbd-2 gene fixedpoint knock-in pigs free from resistant marking can be obtained.

The invention discloses a reconstructed oocyte of a deaf model pig and a construction method of the reconstructed oocyte, and also discloses the deaf model pig and a construction method and application of the deaf model pig. According to the reconstructed oocyte of the deaf model pig, the construction method of the reconstructed oocyte and the construction method of the deaf model pig, by knockingout a pig OSBPL2 gene, the reconstructed oocyte of a deaf mini-pig is obtained, and in combination with a CRISPR / Cas9 gene editing technology, a somatic cell nuclear transfer technology and an embryotransfer technology, high efficiency and feasibility of the construction method for the genetically engineered deaf model pig are proven. Through the construction method of the deaf model pig, genotypes and deafness phenotypes of human OSBPL2 gene mutations can be precisely duplicated, the pig of which an OSBPL2 gene is knocked out has typical characteristics of human autosomal dominant hereditary hearing loss (DFNA67), therefore, a reliable big animal model can be provided for disease research of the human hereditary hearing loss, and the deaf model pig can be applied to inner-ear gene repairing, inner-ear hair cell regeneration, hearing reconstruction and the like.

The invention discloses a culture solution for promoting in-vitro maturation of porcine oocyte, the culture solution takes an M199 culture solution without HEPES as a basic culture solution, and porcine follicular fluid, L-cysteine, sodium pyruvate, epidermal growth factor, insulin, gonadotropin, chorionic gonadotropin and WNT5A cytokine are also added. When the culture solution is used for carrying out in-vitro culture on the porcine oocytes, the in-vitro maturation efficiency and quality of the oocytes can be effectively improved, the obtained mature oocytes are used for carrying out porcinein-vitro fertilization and somatic cell nuclear transfer embryo production, and the blastocyst development rate and quality of the mature oocytes are also remarkably improved.