80 results about "Embryo transplantation" patented technology

The invention discloses a construction method and application of an NMD escape model of an Alport syndrome mouse. A non-human animal model carrying Col4a5 gene c.4432delG frame shift mutation is prepared on the basis of a CRISPR / Cas9 gene editing technology. The method comprises the following steps: co-injecting gRNA of a 49 exon of a targeted Col4a5 gene, homologous recombinant donor oligonucleotide containing c.4432delG mutation and Cas9 nuclease into a mouse fertilized egg, and carrying out embryo transplantation to obtain an F0-generation mutant mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and a stably inherited mutation line is established through two generations of breeding. Through verification, the model accords with pathological characteristics of the Alport syndrome, can stably simulate typical clinical manifestation and pathological characteristics of the human X-linked Alport syndrome, and can be used as an important tool for research of the Alport syndrome.

The invention relates to the technical field of genetic relationship identification, in particular to a construction method, an identification method and application of a primary genetic relationship identification model based on ultralow deep sequencing. According to the identification model obtained by using the construction method of the genetic relationship identification model provided by the invention, the genetic relationship can be identified by adopting ultra-low depth sequencing (0.01 *), the genetic relationship can be determined according to the heterozygosity index between samples to be identified, the prediction accuracy is up to 99.9%, and the construction method is a first-grade genetic relationship detection technology with low cost and high accuracy; the method has the advantages of simple and convenient scheme, high accuracy, high credibility, high repeatability and high economic benefit, has multiple application scenes, and can advance an embryo paternity test window from antenatal / postpartum to before embryo implantation, thereby avoiding physical, mental and economic losses of a patient; ovum / sperm fertilization errors can be found in advance, non-parent embryo transplantation is avoided, and loss caused by embryo wrong transplantation is prevented.

The invention discloses a genetic characteristic index quantitative evaluation method based on embryo genetic information processing and a computer program product, and particularly relates to the technical field of genetic information processing.The method comprises the steps that firstly, whole genome sequencing is conducted on an embryo sample, and pathogenic related mutations are screened through a variation recognition algorithm; then extracting variation points and structural features, and constructing vector description data; further generating two fixed value indexes of pathogenic load and structural stability as model input to calculate an embryo genetic health score; and finally, sorting is performed according to a scoring result, so that the embryo transplantation priority is determined, and scientific and accurate embryo optimization is realized. According to the method, comprehensive identification of embryo genetic states is realized through whole genome sequencing, a pathogenic load and structural stability double-index system is constructed, three-dimensional quality evaluation is realized, and the scientificity, accuracy and individualized clinical application capability of embryo screening are improved by combining a grading result and a family medical history calibration mechanism output by a grading model.

Disclosed are methods for producing controlled endometrial organoids using micropatterning techniques, and methods for using endometrial cells or organoids for diagnostic and predictive purposes, and for optimizing IVF and other infertility-related treatments, including embryo transfer and intrauterine insemination (IUI). These organoids grow in a spatially controlled manner and exhibit less heterogeneity than organoids grown directly in a three-dimensional culture. The specification also describes methods for predicting the success of embryo transfer or other IVF-related treatments, optimizing embryo transfer, improving embryo implantation in the endometrium, and performing embryo transfer in subjects. These methods include culturing endometrial organoids or endometrial epithelial cells, and contacting the endometrial organoids or cells with one or more blast-like cells. In some embodiments, the endometrial organoids or cells include endometrial cells obtained from subjects undergoing or considering IVF-related treatment.

The invention belongs to the technical field of GPCR drug research and development, and discloses a ratio type cAMP fluorescent probe RaCamp and a construction method of a transgenic mouse, the probe RaCamp is formed by series fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein through a flexible linker, and the preparation method of the probe RaCamp comprises the following steps: S1, obtaining an mCherry fragment; s2, a G-Flamp2 linear carrier is obtained; s3, homologous recombination; s4, converting and screening; the construction method of the transgenic mouse comprises the following steps: T1, designing a carrier; t2, microinjection of fertilized eggs; t3, embryo transplantation and reproduction; the invention has the beneficial effects that mCherry is added as an internal reference protein on the premise of keeping the sensitivity of the original G-Flamp2 probe, and the selected connecting peptide (GGGGS) 2 enables the expression quantity of the two proteins in cells to be accurately controlled to be 1: 1, so that the problem of background interference in cell observation of in-vivo imaging is well solved. And the sequence is inserted into a Rosa26-LSL box, so that the construction of a Rosa26-LSL-RaCamp mouse is realized.

The application provides a construction method of a humanized keloid mouse model, comprising the following steps: BAC plasmid construction and preparation; superovulation of experimental mice and collection of zygotes; pronuclear microinjection of zygotes; post-injection embryo transplantation; genotype identification; breeding and genetic analysis; and verification of construction results of the humanized immune system by immunohistochemistry and immunofluorescence. In the application, peripheral blood mononuclear cells treated by sCD27 and skin around keloids (homologous cells and tissues) are transplanted into NSG-MHC-DKO immune-deficient mice with overexpression of CD70 genes, so that HLA rejection of different homologous immune cells and tissues can be avoided, the internal microenvironment of keloids and the interaction between the internal microenvironment and the immune system can be restored to the maximum extent, and the influence of immune factors on the occurrence and development of keloids can be realized in vitro. The application first discovers and verifies that the activation of the CD27-CD70 axis can promote the occurrence and development of keloids, and provides a suitable animal model for the research and development of anti-keloid drugs, especially immunotherapy.

The invention discloses a construction method of a Klf6 gene knockout mouse embryo model, which comprises the following steps: S1, preparing a gene editing solution containing Cas9 enzyme and sgRNA for targeting a third exon region and a fourth exon region of a mouse Klf6 gene by utilizing a CRISPR / Cas9 gene editing technology; s2, taking out the fertilized eggs of the mouse, transfecting the fertilized eggs of the mouse with a gene editing solution, and then performing in-vitro culture on the fertilized eggs; s3, carrying out embryo transplantation on the two-cell stage embryo obtained in the step S2 into a pregnant female mouse, and then carrying out conventional feeding; and S4, 7-8 days after embryo transplantation, taking out the uterus and stripping the embryo to obtain the Klf6 gene knockout mouse embryo model. According to the method, gene knockout is carried out by using CRISPR / Cas9 in the fertilized egg period in combination with an electrotransfection method, and compared with an existing method, the construction process time can be greatly shortened, the working efficiency is obviously improved, and the success rate is high.

The utility model discloses an operating table for fixing small and medium-sized animals. The operating table comprises a table top, a containing groove is formed in the table top, a plurality of fixing piles are arranged on the table top, and the fixing piles are arranged in the circumferential direction of the containing groove; the influence on the positions of small and medium-sized animals in embryo transplantation is avoided, and the influence on embryo transplantation caused by animal fixation in the operation process is avoided.

The present application relates to the technical field of pharmaceutical preparation, in particular to an ultrasonic imaging composite gel, a preparation method and application thereof. The present application provides an ultrasonic imaging composite gel, which comprises a gel matrix, an ultrasonic imaging material and a pharmaceutically acceptable solvent according to mass fraction; the concentration of the gel matrix in the ultrasonic imaging composite gel is 1-500 mg / mL; the concentration of the ultrasonic imaging material in the ultrasonic imaging composite gel is 1-500 mg / mL; the gel matrix comprises one or more of a temperature-sensitive gel matrix, an injectable gel matrix and an ion-responsive gel matrix; the ultrasonic imaging material comprises an ultrasonic phase transition imaging agent and a polymer material wrapping the ultrasonic phase transition imaging agent, and the ultrasonic imaging material is an ultrasonic imaging microbubble structure. The ultrasonic imaging composite gel can enhance ultrasonic imaging in the process of embryo transplantation and promote the adhesion and implantation of embryos and endometrium.

The invention discloses a muscle cell efficient growth gene knock-in cell line construction method based on a CRISPR-Cas9 system, and belongs to the crossing field of gene editing and biotechnology. Through a CRISPR-Cas9 mediated gene targeting insertion technology, fixed-point integration of muscle efficient growth related functional genes is realized in bovine fetal fibroblasts, and a gene overexpression cell line with stable hereditary characteristics is successfully constructed. The cell line can be used as a nuclear donor, and precise creation of high-muscle-production-performance gene editing beef cattle is realized by combining a somatic cell nuclear transfer technology and an embryo transfer technology. The key technical support is provided for beef cattle molecular design breeding by directionally enhancing the cattle muscle development core regulation and control network. The genetic improvement new material cultivated based on the technology is expected to significantly improve the beef cattle muscle growth efficiency and industrial economic benefits, and shows important application value and industrialization prospects in the field of agricultural animal genetic engineering.

An embryo transfer device is provided with a proximal end part forming a proximal end side, an embryo accommodating part, and two or more expansion frame parts configured so as to be brought into a state of approaching each other by elastic deformation when disposed in a tube, and so as to extend out of the tube and be disposed in a uterine cavity, and an X-axis extending in a direction orthogonal to the X-axis passing through the proximal end.

The invention discloses an animal embryo biopsy sampling method and application thereof, and belongs to the technical field of animal breeding. The animal embryo biopsy sampling method provided by the invention is safer, more accurate and more efficient, and can effectively reduce the difficulty of an embryo biopsy sampling technology, reduce mechanical damage to embryos, effectively avoid development retardation of the embryos after sampling and remarkably improve the ratio and utilization efficiency of available embryos on the premise of ensuring the stability of the detection rate of a genetic evaluation chip; the survival rate, the developmental ability and the embryo pregnancy success rate of the sampled embryos are remarkably improved, and the transplantation conception rate of the frozen embryos after sampling reaches 40%; the method can be used for hereditary performance detection and evaluation and embryo screening before embryo implantation, and has important significance in shortening generation intervals, accelerating the breeding process and reducing the overall production cost of animal breeding and breeding.

The invention relates to the technical field of gene engineering, and discloses a method for obtaining smooth hair surface type dairy cow by injecting RNP (Ribonucleic Acid) into cattle fertilized egg cytoplasm, which comprises the following steps: synthesizing sgRNA (Single Guide Ribonucleic Acid); collecting and maturing bovine oocytes; carrying out in-vitro fertilization and early embryo culture; in-vitro embryo microinjection: placing the fertilized ovum in a micromanipulation solution, sucking a mixed solution of WT-Cas9 protein and sgRNA188 by a microinjection needle, carrying out injection, and after the injection is completed, placing the fertilized ovum in BO-IVC liquid drops for continuous culture; embryo transplantation; birth calves are obtained through delivery, and genotype identification is carried out. According to the method for obtaining the smooth hair surface type dairy cow by injecting RNP into the cattle fertilized egg cytoplasm, the mixed solution of sgRNA188 and Cas9 wild type protein is injected into the cattle fertilized egg, the SLICK phenotype Holstein dairy cow is cultivated, and an effective breeding method is provided for breeding of heat-resistant variety cattle.

The invention discloses a repeated implantation failure analysis method based on a multi-modal data fusion model, and the method comprises the steps: collecting basic information of a patient, medical history, laboratory and iconography examination, embryo transplantation and other multi-modal data, integrating dispersed information, providing a comprehensive data basis for analysis, breaking an information island, and enabling the analysis to be based on a complete image; meanwhile, a neural network model is trained according to the preprocessed data to obtain a multi-modal data fusion model, potential correlation among factors can be mined, internal relations can be accurately reflected through fusion features, complex disease causes can be comprehensively revealed, and the limitation that only one aspect can be concerned in the prior art is overcome; besides, a prediction method based on the multi-modal data fusion model is more accurate and reliable than traditional experience judgment and single index analysis, targeted suggestions can be given according to prediction, personalized guidance is provided for clinicians, formulation of precise strategies is assisted, and the defects of precise prediction and personalized suggestions in the prior art are overcome.

The invention discloses a method for promoting in-vitro maturation of sheep oocytes. The composition comprises an induction culture solution A and an induction culture solution B. The induction culture solution A and the induction culture solution B comprise a basic culture solution, and the induction culture solution A further comprises AMH; the induction culture solution B also contains FSH; the basic culture solution comprises an M199 basic culture medium, oestrus sheep serum, sodium pyruvate, luteinizing hormone, estradiol and a penicillin and streptomycin mixed solution. According to the composition and the method, through the synergistic effect of FSH treatment and AMH treatment, the maturation rate, the activity and the development potential of sheep oocytes are remarkably improved, so that the success rate of in-vitro fertilization and embryo transplantation is improved, an important scientific basis is provided for sheep reproduction and assisted reproduction technologies, related technologies are further optimized, and the application prospect is wide. The reproduction success rate is improved.

The invention provides a preparation method of a genetically modified mouse capable of mediating Tbx1 inherent disorder protein structural domain deletion. The method comprises the following steps: (a) constructing a targeting vector; (b) preparing a gene editing compound; (c) microinjection; (d) embryo transplantation and screening; (e) breeding and establishing a stable strain. Sequencing verifies that the gene modified mouse can stably express the Tbx1 mutant protein which lacks IDR and carries an N-terminal 3xFlag tag, and a homozygote mutant mouse shows remarkable developmental defects such as ventricular septal defect and craniofacial deformity; and an important genetic tool is provided for researching the action mechanism of the phase separation characteristic of the Tbx1 protein in the heart, craniofacial development and congenital heart disease (such as DiGeorge syndrome).

The invention relates to the technical field of biology, and discloses a kit for predicting clinical pregnancy outcome of first-time frozen embryo transplantation. The kit comprises immune cells and immune factors in blood, the immune cells comprise CD19 < + > B cells, CD3 < + > T cells, CD4 < + > Th cells, CD8 < + > Ts cells, CD4 < + > / CD8 < + > ratio cells, CD56 < + > NK cells, CD68 < + > monocytes, CD86 < + > M1 sample monocytes, D206 < + > M2 sample monocytes and CD163 < + > M2 sample monocytes; the immune factors comprise IL-1beta, IL-2r, IL-6, IL-8 and TNF (Tumor Necrosis Factor)-alpha. According to the application, the importance of the peripheral blood CD163 + M2 sample mononuclear cell on predicting the continuous pregnancy of first embryo implantation of an FE T patient is confirmed, and the peripheral blood CD163 + M2 sample mononuclear cell is prompted to be possibly a biomarker of an FET period.

The invention belongs to the technical field of embryo biotechnology and embryo transplantation, and particularly relates to unfreezing liquid for bovine vitrification frozen in-vitro embryos and a use method of the unfreezing liquid. The thawing liquid is prepared from the following components: 340 to 515 g / L of trehalose, 0.2 to 0.3 g / L of hyaluronic acid, 6 to 10 g / L of sodium chloride, 0.1 to 0.3 g / L of potassium chloride, 0.05 to 0.15 g / L of magnesium chloride, 0.2 to 0.35 g / L of disodium hydrogen phosphate, 0.1 to 0.3 g / L of monopotassium phosphate, 0.5 to 1.5 g / L of glucose, 0.03 to 0.04 g / L of sodium pyruvate, 0.05 to 0.15 g / L of calcium chloride and 45 to 55 g / L of bovine serum albumin. According to the method, the embryo thawing time can be remarkably shortened while the thawing and transplanting effects are not damaged, the embryo thawing efficiency is improved, the operation process is simple, and popularization and large-scale application of the embryo transplanting technology are facilitated.

The utility model discloses an animal embryo transplantation operating table, and belongs to the technical field of animal embryo transplantation. A groove is formed in the table body, a supporting plate is installed in the groove, four sliding grooves are formed in the top of the table body, the sliding grooves are T-shaped grooves, a fixing assembly comprises a sliding block and a fixing base, a fixing ring is arranged at the bottom of the sliding block, a clamping block is arranged at the bottom of the sliding block, the clamping block is a T-shaped block, the clamping block is clamped in the sliding grooves in a sliding mode, and the fixing base is fixedly connected to the top of the table body. The number of the fixing seats is four, blind holes are formed in the fixing seats, electric push rods are installed in the blind holes, and check blocks are fixedly connected to the moving ends of the electric push rods. According to the operating table, the fixing assembly is arranged, the animal to be operated is fixed on the operating table through the fixing assembly, the situation that the animal struggles in the operation process, and consequently operation is difficult is avoided, and the problem that the animal to be operated cannot be well fixed through the operating table is solved.

The present invention discloses an additive for freezing liquid in a one-step method for bovine in vitro embryos and a one-step freezing method for bovine in vitro embryos, and belongs to the technical field of animal embryo cryopreservation and embryo transplantation. The additive of the present invention is an aqueous solution comprising sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, sodium pyruvate, calcium chloride, bovine serum albumin, sucrose, ethylene glycol and hyaluronic acid; the concentration of sucrose is 4.5 to 5.0 M. Based on the one-step freezing method for bovine in vitro embryos, the present invention increases the sucrose concentration in the conventional one-step freezing liquid and additionally adds hyaluronic acid, thereby increasing the osmotic pressure of the freezing liquid, facilitating the discharge of water inside the embryo, reducing the formation of ice crystals inside the embryo during the freezing process, reducing the damage to the embryo during the freezing process, and improving the survival rate, hatching rate and transplantation pregnancy rate of bovine in vitro embryos after freezing and thawing.

The present invention belongs to the field of embryo biotechnology and embryo transplantation technology, and specifically relates to a thawing solution for bovine vitrified frozen in vitro embryos and a method for using the same. The thawing solution comprises the following components: trehalose 340-515 g / L, hyaluronic acid 0.2-0.3 g / L, sodium chloride 6-10 g / L, potassium chloride 0.1-0.3 g / L, magnesium chloride 0.05-0.15 g / L, disodium hydrogen phosphate 0.2-0.35 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L, glucose 0.5-1.5 g / L, sodium pyruvate 0.03-0.04 g / L, calcium chloride 0.05-0.15 g / L, and bovine serum albumin 45-55 g / L. The present invention can significantly shorten the embryo thawing time without compromising the thawing and transplantation effects, improve the efficiency of embryo thawing, and simplify the operation process, which is conducive to the popularization and large-scale application of embryo transplantation technology.

The utility model discloses an activity preservation device for embryo transplantation, which relates to the technical field of embryo preservation, and comprises a box body, the left part of the bottom of the inner wall of the box body is movably connected with a selection limiting component, and the right part of the upper end of the box body is fixedly provided with a taking component. According to the activity preservation device for embryo transplantation, a selected embryo tube can be accurately and rapidly taken through the selecting and limiting assembly, the output end of the motor can rotate through the rotating disc, the rotating disc rotates the limiting barrel on the rotating disc to the position below the fixing mechanism, and therefore the specified embryo tube can be taken, and the operation of opening a box door is not needed. The specific embryo tube can be taken out of the box body through the taking assembly, other embryo tubes cannot be directly exposed in the air in the taking-out process, and the electric push rod can drive the fixing mechanism to pull out the limiting barrel in the containing groove upwards, so that rapid taking of the single embryo tube is completed, and interference on storage of other embryos can be avoided.

The present invention discloses an embryo transplant gun and a transplant method for sheep breeding, which relate to the technical field of embryo transplantation, comprising: a transplant cylinder, a culture medium tube and a flexible catheter that are coaxially connected in sequence; a push injection assembly that is slidably arranged in the transplant cylinder, and the push injection assembly comprises: a propulsion rod with a predetermined rigidity; a spherical connector fixedly connected to the distal end of the propulsion rod; a piston that is slidably sleeved on the propulsion rod, and an annular groove is provided at one end of the piston away from the spherical connector; a phase change connector, and the phase change connector is embedded in the annular groove through low-temperature phase change to achieve a controllable detachable connection between the propulsion rod and the piston, and the whole has the advantages of bending adaptability, structural stability and sealing.

The invention belongs to the technical field of embryo transplantation, and particularly relates to a cervix dilation device for bovine embryo transplantation, which comprises a handheld part, a dilation part, a wiping mechanism, a liquid supply mechanism and a storage mechanism, the handheld part comprises a mounting shell and a storage box fixedly mounted at the bottom of the mounting shell; the expansion mechanism comprises a supporting pipe, a control wheel and an extrusion wheel, a through hole allowing the supporting pipe to slide is formed in the outer wall of the mounting shell, the extrusion wheel is used for extruding the supporting pipe and elastically arranged in the mounting shell, the control wheel is rotationally mounted in the mounting shell, and the radial outer wall of the control wheel is attached to the outer wall of the supporting pipe; the wiping mechanism comprises a mounting ring and a wiping layer, the mounting ring is of a hollow structure and is rotatably mounted on the inner wall of the mounting shell, and the wiping layer is fixedly mounted on the inner wall of the mounting ring; the liquid supply mechanism is used for providing disinfectant for the inner cavity of the mounting ring; the storage mechanism comprises a storage box and an elastic strip; through cooperation of the structure, the maternal stress reaction is effectively reduced, and the embryo transplantation success rate and the operation safety are improved.

This invention relates to a method for constructing a mouse model of autosomal dominant polycystic kidney disease and its applications, particularly to a method for preparing a non-human animal model carrying a nonsense mutation in the Pkd1 gene c.616_618GAG>TAG based on CRISPR / Cas9 gene editing technology. The method involves co-injecting mouse zygotes with gRNA targeting exon 5 of the Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease. Mutant mice are obtained via embryo transfer. The mutation site is verified by PCR combined with sequencing, and a stable mutant line is established through breeding. This invention also includes the application of this model animal as a research tool for autosomal dominant polycystic kidney disease, particularly in the areas of pathogenesis analysis, drug screening, and therapeutic target validation. This model can stably simulate the typical clinical manifestations and pathological features of human autosomal dominant polycystic kidney disease, providing an important tool for basic research and translational medicine of this disease.

The invention discloses a mammal in-vitro semen optimization method based on charge treatment, and relates to the technical field of livestock breeding biology, and the method comprises the following steps: (a) obtaining mammal semen in an in-vitro environment; (b) putting the sperm into a charge treatment device, and applying a directional electric field to enable the surface of the sperm to carry charges with preset polarity; (c) controlling the electric field intensity to be 5mV and the treatment time to be 30s-60s, so that the sperms obtain the charge attribute opposite to the surface charge of the ovum zona pellucida; (d) directly applying the treated seminal fluid to artificial insemination or in-vitro insemination operation, the technology can improve the success rate that the sperms penetrate through zona pellucida, especially has a breakthrough improvement effect on pathological infertility caused by unbalanced reproductive nutrition of female animals, and is suitable for artificial insemination and embryo transplantation scenes of various mammals at the same time; and high-quality breeding stock resources are efficiently utilized on the premise that the operation convenience is guaranteed.

The invention provides an endometrial receptivity evaluation method, device and equipment, a medium and a program product. The method comprises the following steps: acquiring a fresh embryo transplantation type, clinical data of a patient and a uterus longitudinal section ultrasonic image of an ovum taking day; performing endometrial segmentation on the ultrasonic image to obtain an endometrial region, and determining a peripheral region based on the endometrial region and a transplantation type; extracting radiomics characteristics of the endometrial region and the peripheral region, and extracting clinical characteristics of the clinical data; and inputting the image omics characteristics and the clinical characteristics into an endometrial receptivity multi-modal evaluation model corresponding to the transplantation type to obtain the pregnancy probability output by the multi-modal evaluation model after the fresh embryo transplantation in the current period. According to the method, the device, the equipment, the medium and the program product provided by the invention, real-time analysis is carried out in combination with an artificial intelligence technology, and an evaluation result of the fresh embryo transplantation period can be provided in a short time.

The invention discloses an embryo planting rate prediction method, device, equipment and medium, and relates to the field of assisted reproduction technology and artificial intelligence, the method comprises the following steps: measuring mtDNA copy number and gDNA copy number in a medium after embryo transplantation of a single blastocyst transplantation patient, and constructing a data set; establishing a first model according to the mtDNA copy number and the gDNA copy number in the data set; readjusting the first model by utilizing a bootstrap method to obtain a second model; and predicting the embryo planting rate by using the first model and the second model, so that the problem of reducing the embryo planting rate due to the influence of free gDNA on the integrity and copy number of mtDNA can be solved.

The utility model discloses a puncture needle, relates to the technical field of embryo transplantation, and aims to solve the problem that embryo transplantation cannot be realized when the genital tract of a patient is abnormal by a transplantation tube in the related technology. The puncture needle is used for embryo transplantation and comprises a needle tube, and the needle tube is a rigid tube; the first end face of one end of the needle tube in the length direction is obliquely arranged relative to the axis of the needle tube to form a needle tip, and the needle tip is used for entering a life body; a first included angle between the first end face and the axis of the needle tube is 15-20 degrees.

The invention relates to a method for constructing a porcine endogenous retrovirus inactivated xenotransplantation donor pig, and belongs to the technical field of xenotransplantation. And the copy number of the PERVs and the genotype of the PERV-pol are determined through ddPCR, Sanger sequencing and second-generation PCR deep sequencing. The method comprises the following steps: designing and synthesizing a porcine PERV-pol gene targeting sequence based on a CRISPR / Cas9 RNP system, co-transfecting the porcine PERV-pol gene targeting sequence and spCas9 protein into a porcine fetal fibroblast line, screening to obtain positive cells, carrying out somatic cell cloning and embryo transplantation, taking out a fetus during pregnancy, identifying the PERV-pol knockout condition, and if the PERV-pol knockout condition is completely carried out, carrying out somatic cell cloning and embryo transplantation; and if the PERV-pol gene is not completely knocked out, secondarily designing and synthesizing an sgRNA targeting sequence, transfecting the sgRNA targeting sequence into the fetal fibroblast cell line, screening to obtain a cell line in which the PERV-pol gene is completely knocked out, and carrying out somatic cell cloning and embryo transplantation to obtain the PERVs inactivated xenotransplantation donor pig. The problem that the PERVs inactivation efficiency is low is solved, and the PERVs-inactivated xenotransplantation donor pig can be obtained through one round or multiple rounds.