42 results about "Microinjection" patented technology

The invention relates to the technical field of agricultural biology, in particular to application of a neuropeptide gene CpILP6 in relieving diapause of codling moths. After siRNA is microinjected to silence CpILP6 in a targeted manner, the diapause state of CpILP6 is changed and tends to a normal development phenotype; the research result of the invention provides a theoretical basis for defining the regulation function of the neuropeptide gene CpILP6 in the diapause induction and maintenance process of codling moth.

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.

Provided herein are methods, computer programs, and systems for automated microinjection, for example, automated Intracytoplasmic Sperm Injection (ICSI), wherein creating, a first dataset of an oocyte and a holding device and a second dataset of an injection pipette; detecting the oocyte and the holding device in the first dataset and the injection pipette; selecting the image of the first dataset and of the second dataset where an equatorial plane of the oocyte / holding device and of the injection pipette has an improved focusing parameter; selecting images of the first and second datasets and labeling the pixels associated with the oocyte and to the injection pipette; creating an injection trajectory for the injection pipette to perform the ICSI using detected morphological structures; detecting when the oocyte is rupturing and when the spermatozoa has been released from the injection pipette into the cytoplasm of oocyte.

The invention belongs to the technical field of transgenic and biomedical research tools, and particularly relates to a method for constructing a transgenic fish system based on a Tol2 transposon system, which comprises the following steps: constructing a myo6b-lhfpl5b-DsRed plasmid by using myo6b as a promoter, lhfpl5b as a target gene and DsRed as a red fluorescent protein tag through a microinjection method, and constructing a Tol2 transposon system-based transgenic fish system. Co-injecting the plasmid containing the Tol2 transposase mRNA and the Tol2 transposase mRNA into a fertilized egg of an AB zebra fish strain, and screening generation by generation to obtain a transgenic fish strain with stable inheritance MET channel gene lhfpl5b marked hair cells. The spatio-temporal dynamic visualization of the living whole fish to the lhfpl5b is realized, the deafness-causing molecular pathway is clarified, and an in-vivo platform is provided for drug screening and intervention strategies.

The application belongs to the technical field of biology and particularly relates to a functional recombinant Cas9 protein targeted to an ovocyte of Procambarus clarkii and application thereof. The protein is NLs-VgSP-Cas9-NLs. The application directly delivers the recombinant Cas9 protein, thereby reducing the risk of integration of exogenous genes. By specifically targeting the ovocyte, off-target effects on somatic cells can be avoided, the efficiency of editing of germ cells is improved, and early development or genetic manipulation can be conveniently studied. The ovocyte of Procambarus clarkii is large, the recombinant Cas9 protein is efficiently delivered by receptor-mediated endocytosis on the surface of the ovocyte, and the problem that microinjection of zygotes cannot be implemented is solved.

The invention belongs to the technical field of animal reproductive biology and cell engineering, and particularly relates to a fluorescence labeling expression system for visualizing an oocyte nucleus maturation process and application of the fluorescence labeling expression system. The fluorescence labeling expression system comprises an eukaryotic expression vector pVenus-LMNB1; the eukaryotic expression vector pVenus-LMNB1 comprises a nucleotide sequence for coding the fusion protein Venus-LMNB1. According to the invention, a nuclear fiber layer protein stably expressed on a nuclear membrane is selected as a research object, a eukaryotic expression vector pVenus-LMNB1 is constructed by using a molecular cloning technology, an LMNB1 gene containing a green fluorescent label Venus is overexpressed in a porcine oocyte by using a microinjection technology, fluorescent protein labeling of a structural protein on the nuclear membrane is utilized, and the expression vector pVenus-LMNB1 is constructed. The expression positioning condition of the LMNB1 in the maturation process of the porcine oocytes is observed, and a powerful tool is provided for development of in-vitro culture related researches of the porcine oocytes.

The present application relates to the field of biotechnology, and more particularly to a method for knocking out runx2b gene of heterologous octoploid Changfeng crucian and application thereof. The present application designs gRNA-1 and gRNA-2 aiming at all the alleles of runx2b from two sets of parent sources of heterozygous gynogenetic crucian and Xingguo red common carp in Changfeng crucian; the mixture of two gRNAs synthesized by in vitro transcription and Cas9 protein is introduced into the animal pole of single-cell stage of Changfeng crucian fertilized eggs by microinjection, which can efficiently knock out all the 8 alleles of runx2b-A and runx2b-B. Any gRNA can guide the CRISPR / Cas9 system to simultaneously edit the 8 alleles; gRNA-1 achieves 97.5% of synchronous knockout rate, and gRNA-2 achieves 95% of synchronous knockout efficiency; when the double gRNA collaborative cutting strategy is used, the complete editing efficiency of the 8 functional copies reaches 100%.

The invention discloses application of transgenic zebrafish in preparation of an animal model capable of visually monitoring mast cells. The transgenic zebrafish is prepared by the following method: respectively carrying out XhoI and BamHI double enzyme digestion on a cpa5 gene promoter sequence (SEQ ID NO: 1) and plasmids with fluorescent protein genes and Tol2 transposon recognition sites, and then connecting to obtain recombinant plasmids; and then introducing the recombinant plasmid and Tol2 transposase mRNA into a zebra fish embryo in a microinjection mode in a 1-cell period of zebra fish embryo development, and culturing at constant temperature to obtain the transgenic zebra fish. The cpa5 promoter is constructed to drive an expression framework of the fluorescent protein, so that the mast cells naturally emit fluorescence in the zebra fish body, and an effective animal model is provided for mechanism research and drug screening of mast cell related diseases.

The invention belongs to the technical field of bioengineering and analytical chemistry, and provides a probe eZinCh-2 modified plasmid based on FRET (Fluorescence Resonance Energy Transfer) and application of the probe eZinCh-2 modified plasmid in zinc ion detection. According to the invention, the plasmid is constructed through molecular cloning and homologous recombination technologies, and is microinjected into a zebrafish embryo, so that a transgenic strain capable of stably inheriting and expressing the probe protein is successfully obtained; as a living body biological tool, the zebrafish of the strain is combined with a confocal microscope spectrum FRET technology, and real-time and quantitative monitoring and visual imaging of dynamic changes of zinc ions at living body, cell and tissue levels are achieved. The method solves the problem that real-time dynamic visual monitoring of the zinc ions cannot be realized in a living body model in the prior art, and is suitable for real-time microscopic quantitative analysis of the content of the zinc ions in model organisms such as zebra fish and the like.

This invention provides a research model and construction method for calcium ion imaging of ASH neurons in nematodes, belonging to the field of biological model construction technology. The construction method includes the following steps: nematode culture; construction of a transgenic probe plasmid: obtaining the ASH neuron-specific promoter sra-6 using PCR technology, and connecting HindIII and BamHI restriction sites to both ends of the promoter; amplifying YC3.60 using PCR technology; then inserting the promoter sequence into the pPD95.75 plasmid, and then inserting YC3.60 after the promoter. Transgenic microinjection of nematodes is used, followed by screening to obtain nematodes with ASH neurons carrying green fluorescent protein. In this application, YC3.60 is transferred into nematodes via transgenic means. Using a promoter specifically expressed in ASH neurons to link the gene of this fluorescent probe protein into the nematode, the fluorescent probe can be specifically expressed in the neuron. The constructed nematode model can be used for neuronal structure and functional imaging studies, as well as neuropharmacological efficacy analysis.

The invention discloses a non-human animal integrated with a double-derived VH subclass fusion gene as well as a construction method and application of the non-human animal. The method comprises the following steps: firstly, constructing a gene construct containing human IGHV1-69 and IGHV3-23 fragments, a specific spacer region, a functional element and a rat heavy chain 3'enhancer, and microinjecting the gene construct into a C57BL / 6J fertilized egg with an endogenous heavy chain and a kappa light chain knocked out to obtain a transgenic mouse; through identification, after a mouse endogenous antibody is inactivated, a human gene is stably integrated, a human IgM antibody is expressed and H5N1 HA immunization is carried out, the use frequency of IGHV1-69 is remarkably increased, the mouse produces a high-titer specific antibody, and serum has neutralizing activity. The mouse can generate a target bnAbs aiming at the HA stem and the RBS in parallel, so that the antibody screening efficiency is improved, and an in-vivo screening and evaluation platform for simulating human body fluid response is provided for research and development of influenza antibodies and vaccines.

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 discloses a construction method and application of an Ace2 gene modified golden hamster model, and belongs to the technical field of genetic engineering animal model construction. According to the invention, sgRNA specifically targeting a golden hamster Ace2 gene is prepared, the golden hamster Ace2 gene is accurately targeted by using a two-cell microinjection technology, and gene modification is realized, so that a gene modified hamster is obtained. According to the invention, the golden hamster is used as a model background animal to construct an SARS-CoV-2 research and application tool mouse, and the constructed Ace2-deficient golden hamster model is applied to the field of research and application of a novel coronavirus disease model.

The invention discloses a low-density sperm braking method without a PVP medium in ICSI fertilization, and relates to the field of assisted reproduction therapy, and the method comprises the following steps: step 1, diluting a sperm suspension until the density is 0.1 * 10 < 6 > / ml, and placing the diluted sperm suspension in an in-vitro operating solution without PVP; step 2, under an inverted microscope, directly pressing and braking the tail of the sperm in the in-vitro operating fluid by using a microinjection needle, so that the tail of the sperm is folded or curled to deform; and step 3, transferring the braked sperms into operation liquid drops containing ovum to complete microinjection, and performing tail pressing operation in an isotonic operation liquid without PVP (Polyvinyl Pyrrolidone), so that the tail parts of the sperms present visible curled deformation or tail folding. The sperm motility destruction rate is 100% (residual motility is completely prevented from interfering fertilization), and the tail cell membrane rupture rate is 100% (it is ensured that activation factors in sperms are fully released).

This invention relates to the field of gene editing technology, and more particularly to a method for constructing a zebrafish epilepsy model with htr6 gene deletion. Using CRISPANT technology, this invention designs suitable target sites on the htr6 gene in zebrafish, and co-injects specific sgRNA synthesized in vitro with Cas9 protein into zebrafish fertilized eggs via microinjection, successfully constructing a zebrafish epilepsy model. This invention can more efficiently and accurately silence specific genes in the genome of an organism, and is simple and low-cost to produce. It can also simultaneously cleave multiple sites on the target gene, silencing any number of individual genes. The zebrafish epilepsy model provided by this invention lays a solid foundation for further research on the relationship between htr6 gene mutations and the pathogenesis of epilepsy, as well as for screening antiepileptic drugs.

The application discloses a method for constructing a fluorescently labeled piwil1 gene knockout sterile zebrafish line, which utilizes a CRISPR / Cas9 system to construct a piwil1 gene knock-in vector carrying P2A-EGFP or P2A-mCherry, respectively, to obtain two hybrid strains through microinjection, and then to screen a homozygote expressing EGFP and mCherry simultaneously by mating the two strains. The homozygote shows male sterility and can be used for reproductive development research and ecological prevention and control of transgenic fish. The application realizes visual labeling of the function of the piwil1 gene and accurate screening of the sterile phenotype, and is simple and efficient to operate.

The invention relates to the technical field of gene engineering, in particular to a Cilp gene enhancer and application thereof. Through integration and verification of an ATAC-seq map, a single cell transcriptome data set and multi-omics data, the Cilp gene enhancer is obtained through screening, and a mouse inner ear microinjection system is utilized to further prove that the Cilp gene enhancer can significantly enhance the transcriptional activity of an EGFP protein gene in cochlea type 2 neuronal cells. Compared with other sequences with similar sizes, the Cilp gene enhancer provided by the invention can drive specific expression of a reporter gene or a Cilp gene in cochlear neurons, especially type 2 neurons, and the gene expression level is remarkably improved. The Cilp gene enhancer provided by the invention is suitable for biological materials such as recombinant DNA (deoxyribonucleic acid), vectors or adenoviruses, can be used for promoting transcription of EGFP (enhanced green fluorescent protein) genes or other genes in cochlea neurons, and provides a new targeting tool for gene therapy of hereditary hearing loss.

The invention discloses a rat and mouse laparoscope operating system which comprises a supporting body, an imaging assembly and an operating assembly, the imaging assembly is connected to the supporting body, electrically connected with external display equipment and used for collecting and transmitting image information, the operating assembly comprises functional parts and a driving part, and the functional parts comprise tweezers, scissors and an injection needle. The tweezers, the scissors and the injection needle are detachably connected with the supporting body, and the driving part is connected to the supporting body, can be in transmission connection with the tweezers and the scissors and is used for driving the tweezers and the scissors to move relative to the supporting body. The visual laparoscope micro-injection system can effectively solve the problem that in the prior art, a visual laparoscope micro-injection system which is suitable for rats and mice, can be independently operated by a single person and has the functions of abdominal cavity micro-injection and surgical operation is lacked.

The utility model provides a microfluidic chip for skin cell sorting, which relates to the technical field of cell sorting and comprises a substrate, a microfluidic channel is arranged on the upper surface of the substrate, and the microfluidic channel comprises a primary sorting channel and two secondary sorting channels. The first-stage sorting channel is composed of three sorting matrixes, the second-stage sorting channel is composed of two sorting matrixes, and each sorting matrix is formed by connecting an expansion area with a contraction area. According to the skin cell sorting device, an external magnetic field acts on the immunomagnetic beads and combined cells, so that the sorting efficiency is greatly improved under the condition of effectively sorting the skin cells. Moreover, corollary equipment is simplified and only relates to the upright microscope and the micro-injection pump, so that the operation of the micro-fluidic chip is simpler and more convenient, and the use cost is remarkably reduced.

The invention discloses a crucian egg dispersing, fixing and micro-injection device, and relates to the technical field of fish egg micro-injection, the crucian egg dispersing, fixing and micro-injection device comprises a base and a micro-injection instrument, the upper end of the base is provided with a box body, the box body is internally provided with a partition plate, the partition plate divides the interior of the box body into a collecting cavity and a water collecting cavity, a top plate is arranged above the box body, and the top plate is provided with a water outlet; a top plate is arranged in the box body, a first electric telescopic rod is arranged at the upper end of the top plate, the telescopic end of the first electric telescopic rod penetrates through the top plate and is connected with a microinjection instrument, the rotary table is arranged in the box body, four device grooves are formed in the side wall of the rotary table, and the four device grooves are formed in the circumferential direction of the rotary table at equal intervals; and the four roe dispersing assemblies are arranged on the inner sides of the four device grooves correspondingly. The device can continuously and automatically disperse and fix crucian eggs, perform micro-injection and discharge and collect the crucian eggs, so that the working efficiency of micro-injection of the crucian eggs is greatly improved.

The present application belongs to the technical field of gene editing, and particularly relates to a gene editing method for obtaining a bivalve adult editing body. Specifically, a target gene with an outward visible phenotype and an early development not lethal is taken as a marker, target sites sgRNAs are designed based on the marker, and then a Cas9 / sgRNAs editing body is constructed by means of an electrotransfection system, so that gene editing of the bivalve is realized and the adult editing body is obtained. The present application breaks through the technical bottleneck that the existing gene editing of the bivalve is limited to the larva development stage and the editing body is difficult to screen in adulthood, and provides a new technical system for creating the editing body germplasm of the bivalve. In addition to being applicable to oysters, the technical system is also applicable to other bivalves with small fertilized eggs, difficult microinjection and high larva culture mortality.

This invention relates to an animal model of mediastinal lymph node metastasis in lung cancer and its construction method. The core of this invention lies in the precise intrapulmonary in situ microinjection technique to directly implant human lung cancer cells into the lung parenchyma of immunodeficient mice. Utilizing the natural lymphatic drainage pathway of the lung tissue, tumor cells are induced to migrate to the mediastinal lymph nodes, thus successfully constructing a complete metastasis chain model from the primary tumor to regional lymph nodes. This model can more realistically simulate the microenvironment and process of lung cancer lymph node metastasis, and is suitable for metastasis mechanism research, drug evaluation, and the development of personalized treatment strategies, possessing high translational medicine value.

The invention relates to the technical field of biological medicine, and discloses a method for constructing a Myh6-CRE transgenic golden hamster model and application of the Myh6-CRE transgenic golden hamster model. The invention provides a specific DNA (deoxyribonucleic acid) molecule. The specific DNA molecule sequentially comprises an upstream insulator, a Myh6 promoter, a Cre recombinase coding gene, a beta-globin polyA and a downstream insulator from upstream to downstream. The invention also provides a method for preparing the transgenic golden hamster, which comprises the following steps: introducing the expression construct containing the specific DNA molecule into a golden hamster fertilized egg in a fertilized egg microinjection manner, and randomly integrating the expression construct into a golden hamster genome, thereby obtaining the transgenic golden hamster. The invention further provides a Myh6-CRE transgenic golden hamster model, the transgenic golden hamster expresses Cre recombinase under the driving of a Myh6 promoter, and specific recombination between LoxP sites can be mediated. The Myh6-CRE golden hamster constructed by the invention can be used for establishing a conditional gene knockout model, and is suitable for researching the function and action mechanism of the Myh6 related gene in the occurrence and development process of heart and related diseases.

This invention discloses a method for heavy ion beam mutagenesis breeding of poultry. To address the challenges of efficient, rapid, and safe breeding in poultry, this method utilizes heavy ion beam irradiation of poultry progenitor cells (PGCs) to induce radiation mutagenesis, constructing a mutant cell library. The PGCs in this library are then isolated into single cells to form monoclonal populations. Targeted deep sequencing is used to screen for positive PGCs with gene mutations from the S2-after monoclonal population, and the mutated gene is preliminarily identified. Positive PGCs are injected via microinjection into recipient embryos after their own PGCs have been ablated, resulting in chimeras. Individuals developed from these chimeras undergo molecular screening and identification. M1 generation individuals containing the identified mutated gene are then used for breeding to obtain offspring strains that stably inherit the mutated gene. This method is efficient, rapid, safe, and avoids the ethical and regulatory risks associated with gene editing technologies, making it of significant application value for mutagenesis breeding in poultry.

The application relates to a construction method of a conditional overexpression ZFP595 transgenic mouse model and application thereof, and relates to the technical field of transgenesis.The construction method comprises the following steps: constructing a homologous recombination vector containing a ZFP595 gene, wherein the vector comprises a 3.3 kb 5' homologous arm, CAG-LSL-Zfp595-HA-IRES-tdTomato-Wpre-pA and a 3.3 kb 3' homologous arm; microinjecting Cas9 mRNA, gRNA and the homologous recombination vector into a zygote of a recipient mouse to obtain F0 generation mice, and performing identification; mating the F0 generation mice identified as positive with wild type mice and backcrossing to obtain a mouse model for conditionally overexpressing ZFP595.The conditional overexpression ZFP595 mouse model constructed by the construction method can be crossed with various Cre mice to realize specific overexpression in different tissues, organs and cells.

This invention discloses a method for constructing a mouse model of COL4A5-K229X point mutation X-linked Alport syndrome. Cas9 mRNA, gRNA, and a donor vector were designed and prepared targeting the c.685A>T point mutation in exon 12 of the mouse COL4A5 gene. These components were mixed and microinjected into C57BL / 6J mouse zygotes to obtain F0 generation mice. Genotypes were identified by PCR amplification and Sanger sequencing, and positive mice were screened. Positive F0 generation mice were mated with wild-type mice to breed F1 and subsequent generations. The model phenotype was further verified by qPCR, biochemical analysis, light microscopy, transmission electron microscopy, and immunofluorescence. The model constructed by this invention exhibits hematuria, proteinuria, azotemia, podocyte loss, and irregular thickening and stratification of the glomerular basement membrane, consistent with the phenotype of human XLAS patients, providing an animal model tool for elucidating the pathogenesis and developing treatment strategies.

This invention discloses a method for constructing and applying an anti-obesity animal model based on ISCA1 / 2 gene regulation, belonging to the field of biomedical technology. The method utilizes CRISPR-Cas9 gene editing technology to design specific single-stranded guide RNAs targeting the ISCA1 and ISCA2 genes in mice. Cas9 mRNA and sgRNA are introduced into fertilized eggs of C57BL / 6J strain mice via microinjection. The injected embryos are then transferred into pseudopregnant mice to obtain F0 generation chimeric mice. By mating F0 generation mice with wild-type C57BL / 6J mice, F1 generation heterozygous mice carrying the target mutation in the germline are selected, thus obtaining the anti-obesity animal model. This model is not only a phenotypic model but also a mechanistic research model, suitable for drug target validation, energy metabolism pathway exploration, and personalized treatment strategy evaluation.

The invention discloses a method for rapidly constructing a Foxn1 homozygous nude mouse through RepSox induction based on CRISPR / Cas9, and the method comprises the following steps: synthesizing two sgRNAs for respectively identifying a 5'terminal target site and a 3 'terminal target site, the nucleotide sequence of the sgRNA for identifying the 5' terminal target site is as shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA for identifying the 3 'terminal target site is as shown in SEQ ID NO: 2; a mixed solution of the two sgRNAs, the Cas protein and the RepSox is microinjected into the fertilized eggs of the rats; the injected embryos are subjected to transient in-vitro culture, survival embryos are screened and transplanted into fallopian tubes or uteruses of pseudopregnant female rats for development, and F0-generation Foxn1 knockout homozygous rats are obtained. According to the method, the Foxn1 gene can be accurately knocked out, the survival rate of a gene editing embryo is improved, the model construction period is shortened, and the rat knockout efficiency is remarkably improved.

The application belongs to the technical field of biology, and particularly relates to a construction method of a gnaq knockout strain of rare gobiorynchus and application thereof. The application adopts CRISPR / Cas9 gene editing technology, designs double-target sgRNA (target point 1 induces 8 bp deletion, and target point 2 mediates 32 bp insertion) aiming at gnaq gene of the rare gobiorynchus. The sgRNA sequence is optimized by ZiFiT target point design software, and NCBI BLAST comparison is performed to ensure the targeting specificity. The in-vitro transcribed sgRNA and Cas9 mRNA are microinjected into the fertilized eggs of the rare gobiorynchus to obtain F0 chimeras. After the F1 heterozygotes are self-crossed, the homozygous mutants gnaq-8 and gnaq+32 which conform to Mendelian genetic law are successfully screened in the F2 generation. The gnaq knockout is realized in the rare gobiorynchus for the first time, which fills the gap of the research on G protein signaling pathway of the rare gobiorynchus, and provides an important model for the subsequent research on the functions of related genes of fish (such as antiviral immunity and tumor mechanism).

The application discloses a pig chimeric embryo culture medium and application thereof in effectively improving in-vitro chimeric ability of pig embryo stem cells, and belongs to the technical field of bioengineering.The pig chimeric embryo culture medium is characterized in that growth factors, nutrients and other key components in the culture medium are accurately controlled, so that a neutral culture environment for promoting synchronous development of stem cells and embryos is created.On this basis, the application combines overexpression of anti-apoptotic factors, optimization of microinjection conditions and adoption of laser-assisted hatching means, so as to further improve chimeric efficiency, effectively support integration of stem cells and embryos and improve chimeric efficiency, thereby providing a reliable in-vitro tool for evaluating chimeric ability and pluripotency of pig stem cells.