74results about How to "Improve cloning efficiency" patented technology

The invention relates to methods for culturing human embryonic stem cells by culturing the stem cells in an environment essentially free of mammalian fetal serum and in a stem cell culture medium including amino acids, vitamins, salts, minerals, transferring, insulin, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer the medium. Also disclosed are compositions capable of supporting the culture and proliferation of human embryonic stem cells without the need for feeder cells or for exposure of the medium to feeder cells.

Methods and composition of induction of pluripotent stem cells are disclosed. For example, in certain aspects methods for generating essentially vector-free induced pluripotent stem cells with cell signaling regulators are described. Furthermore, certain aspects of the invention provide novel compositions comprising induced pluripotent stem cells essentially free of exogenous retroviral vector elements in the presence of a medium comprising signaling inhibitors. In certain aspects, feeder-free episomal reprogramming methods may be provided.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system and application thereof to construction of swine-derived recombinant cells with insulin receptor substrate gene defects. The invention provides an sgRNA (ribonucleic acid) combination which is composed of sgRNAIRS1-1, sgRNAIRS1-3, sgRNAIRS2-2 and sgRNAIRS2-3. The invention provides a plasmid combination. The plasmid combination is composed of four plasmids, and the four plasmids are respectively subjected to transcription to obtain sgRNAIRS1-1, sgRNAIRS1-3, sgRNAIRS2-2 and sgRNAIRS2-3. Theapplication of the sgRNA combination or the plasmid combination is as follows: preparation of recombinant cells; preparation of a diabetic cell model; preparation of a diabetic animal model. Accordingto the invention, sgRNAIRS1-1 is as shown in SEQ ID NO:8; sgRNAIRS1-3 is as shown in SEQ ID NO:10; sgRNAIRS2-2 is shown as SEQ ID NO:14; and sgRNAIRS2-3 is as shown in SEQ ID NO:15. A solid foundation is laid for the preparation of diabetic pig models, and important application value for the research and development of diabetic medicaments is achieved.

The invention discloses a method for improving the pig cloning efficiency on the basis of inhibition of H3K9me3 methylation. According to the method, H3K9me3 methylation is inhibited by inhibiting expression of Suv39H1 and Suv39H2 genes in nuclear donor cells during nuclear transplantation of a cloned embryo of a pig and overexpression of KDM4A mRNA during nuclear transplantation of a reconstructed embryo, and the pig cloning efficiency is further improved. Specifically, according to the method for improving the pig cloning efficiency, effective siRNA resisting Suv39H1 and Suv39H2 genes is co-transfected in the nuclear donor cells before nuclear transplantation, overexpression of the Suv39H1 and Suv39H2 genes in the nuclear donor cells is inhibited, accordingly, the high methylation level of H3K9me3 in the nuclear donor cells is inhibited, the processed nuclear donor cells are subjected to nuclear transplantation, cytoplasm microinjection of KDM4A mRNA is performed after the reconstructed embryo obtained after nuclear transplantation is activated, methylation of histone H3K9me3 in the reconstructed embryo is further inhibited, and the cloning efficiency of the pig is finally improved.

The invention provides application of DNA exonuclease to DNA recombinant seamless cloning and provides a kit capable of being used for DNA recombinant seamless cloning. The DNA exonuclease is T5 exonuclease, T7 exonuclease, exonuclease III or Lambda exonuclease or a mixture thereof. The invention further provides a seamless cloning method applying the DNA exonuclease, which comprises the followingsteps: linearizing a vector through a PCR method or restriction endonuclease digestion; introducing homologous sequences that are respectively homologous to the two ends of the vector at the two endsof a target gene fragment through PCR to obtain an amplified target gene fragment; after the treated target fragment and the treated linearized vector are mixed, adding the DNA exonuclease and a reaction solution for temperature bath to obtain a vector and fragment mixture; converting an echerichia coli receptive cell by using the obtained vector and fragment mixture. The application and the method provided by the invention have the following advantages that the site selection can be flexibly performed, and the gene cloning can be performed in any position of the vector; the vector construction can be quickly, simply and conveniently completed within 10 minutes; meanwhile, the cloning is accurate and efficient.

The present invention relates to the DNA sequence for constituting T carrier, carrier containing the said sequence and the method of T carrier conversion. The DNA sequence GGTGACCANNNNT/NNNNNTGG is recognition sequence concatemer of limitative endoenzyme HphI and XcmI. The said two sequences are inserted into carrier without XcmI recognition site in reverse repeated mode to obtain one pre-I carrier. In the presence of competitive exoenzyme substrate in the concentration of 10 moles higher than pre-T carrier, the pre-T carrier is overdigested by endoenzyme XcmI to obtain mature T carrier. Cloning test shows that the T carrier has high cloning efficiency 95% or higher. The said method is simple and has universal application value.

The invention relates to a method for high-flux rapidly cloning of a rape draught-resistant gene. The method comprises the following steps: (1) performing drought stress treatment on a germinal rape seedling, and sampling the whole stalk to obtain a drought stress responded total RNA base; (2) obtaining mRNA and performing inverse transcription to synthesize the double chain cDNA, and constructing a drought stress transcriptome overall length cDNA homogenization library; (3) constructing a drought stress transcriptome overall length cDNA plant expression library; (4) constructing an over-expression rape drought stress transcriptome overall length cDNA arabidopsis mutant library; (5) screening the obtained arabidopsis mutant library by using a high-flux far-infrared thermal imagery technology, and performing drought stress phenotype identification to obtain an over-expression rape drought stress transcriptome arabidopsis transgenic plant; and (6) cloning a rape drought stress response key gene causing the drought stress phenotype from the obtained drought stress arabidopsis transgenic plant. The method is rapid and convenient and the cloning efficiency of the plant drought stress gene is increased.

The invention discloses a yeast-Agrobacterium shuttle vector, and a construction method and an application thereof, and belongs to the field of plant viruses. The shuttle vector simultaneously contains elements and a selective marker which can be stably replicated and expressed in Agrobacterium and yeast, so a virus infectious clone can be constructed by using the high-efficiency homologous recombination system of the yeast, and plant infection is completed by using the Agrobacterium binary vector containing the elements needed by the maintenance and transformation of plant cells and insertionof the plant in the Agrobacterium. The virus infection clone of a large genome or a complex genome can be constructed within two weeks the vector and the construction method. The vector can quickly and accurately construct the virus infection clone, so the yeast-Agrobacterium shuttle vector for rapid construction of the virus infection clone, and the application method thereof have very importantapplication values.

The one aspect of the present invention aims to provide a novel cloning method for human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), which method is based on culture of dispersed single cells without addition of an apoptotic agent. More specifically, the one aspect of the present invention aims to provide a cloning method for hESCs and hiPSCs based on culture of dispersed single cells utilizing shear stress. The above problem is solved by providing a method for culturing dispersed single pluripotent cells, wherein the dispersed single cells are cultured under laminar flow conditions.

The invention is a new phage mid display vector pCANTAB5L, applied to the phage for displaying the exotic random polypeptide. It inserts the linker fragment Xba I-Stu I-Sal I-Kpn I-[G4S] 3-Not I between SfiI and NotI endonuclease cutting sites of pCANTAB5L to obtain the vector. The vector provides 5 common endonuclease cloning sites Sfi I, Xba I, Stu I, Sal I and Kpn I, which makes exotic gene oriented cloning convenient and improves the cloning efficiency, beneficial to enlarge the polypeptide library capacity displayed by the phage; the vector introduces flexible polypeptide linker gene [G4S] 3 composed of 15amino acids between the phage PIII protein gene and the exotic display protein gene, and properly uses Escherichia coli rare codon, so as to reduce the mutual interference of the displayed exotic and PIII proteins and maintain the function and activity of the displayed exotic and PIII proteins. It is applied to displaying various random peptide library, functional target protein, functional target protein variant library and large molecular-weight (>300 amino acids) functional protein.

The invention provides a method for cloning high-flux directed T vector. The method is as follows: an appropriate IIS type restriction enzyme site and lactose operator gene sequence (LacO) are selected; through reconstructing integrate lactose operator gene sequence (LacO) between vector mutagenesis cleavage joint transformation and cloning fragment, directed cloning and non-identification cloning are realized; moreover, fusion and non-fusion T vector construction is realized. The method has simple primer design, high cloning efficiency, less time consumption and low cost; moreover, the obtained recon does not need subsequent identification and is compatible with the prior cloning method.

The invention discloses a preparation method and application of low-temperature bacterium DNA ligase. The prepared low-temperature bacterium DNA ligase has the advantages that the optimal temperature range of the enzyme activity of the low-temperature bacterium DNA ligase is 4-20 DEG C, ligation products of the DNA ligase which has reaction for 5 minutes in the temperature range are larger than 90%, and the DNA ligase is quite suitable for the low-temperature DNA ligation reaction in gene cloning; due to the fact that the DNA ligation reaction of the gene cloning is low in temperature, the optimal temperature of traditional T4 phage DNA ligase is 30-40 DEG C and the optimal temperature of the traditional T4 phage DNA ligase is not matched with the temperature of the DNA ligation reaction of the gene cloning, DNA ligation efficiency is low, long-time reaction is needed, ligation time reaches up to 12 hours, and gene cloning speed is limited greatly; the DNA ligase has the highest catalytic activity under low temperature, the DNA ligation reaction time is shortened greatly, and gene cloning speed is increased.

The invention discloses a cloning method of the animal somatic cells. The cloning method of the animal somatic cells is that tetraploid blastomere in 8-16 cell stage which is obtained from vitro fertilization is transferred into a somatic cloned embryo in 8-16 cell stage to get a compound embryo; the compound embryo is cultured to get a cloned animal. The invention initiatively changes the sourceof trophoblast cell; the tetraploid blastomere obtained from vitro fertilization is put into the somatic cloned embryo in 8-16 cell stage, which can be developed into trophoblast cell; then the placenta quality is improved, the development condition for the fetus is promoted, the pregnancy rate of the cloned embryo is increased, the abortion rate and the dystocia rate is decreased and the cloningefficiency of the somatic cell is greatly improved. As the fetus is constituted by ploid cells, the tetraploid blastomere can only develop into the placenta without the possibility of developing intofetus. Therefore, the method does not affect the individual cloned animal.

The present invention belongs to the field of bioengineering technology, and is especially biological consubstantial cell nucleus grafting and cloning technology. By means of biological cloning technology, the present invention takes consubstantial nucleus donor cell and denucleated egg mother cell with clear genetic background for consubstantial cell nucleus grafting to prepare cloned animal. The present invention has relatively high matching between the donor nucleus DNA and acceptor cytoplasm and the electronic compatibility inside nuclear substance, and can improve the development of cloned embryo and raise cloning efficiency. The said technology may be used in cloning animal to obtain high quality livestock.

The present invention discloses the method of finding antigen determinant with tumor specificity, shown by HLA acceptor and identified by CD4 positive lymphocyte or CD8 positive lymph cell from the tumor sample and tumor penetrating lymphocyte gathered from patient body. The present invention discloses also the method of screening the latent curative effect of treating vaccine or immunogen in cell immunizing therapy on patient of specific race. The present invention discloses also the method of extracorporeal amplifying to generate T-lymphocyte with specific reaction on antigen and antigen determinant for cell transferring therapy or developing treating vaccine to activate cell immunity system.

The invention discloses a method for preparing for T carrier used in T-A clone, which chooses a normal plasmid with high copy quantity as table to design four pairs of primer and uses point discontinuity to construct two types of differential discretion plasmid in the order advance sequence; it separately constructs TTTTAAA and TTTAAAA sequence box on the polyclonal point of the two types of discretion plasmid and leads two different enzyme cutting points BamHI and HindIII; it separately leads the discretion plasmids in the bacteria to augment and uses the inner cutting enzyme of the identifying TTTAAA sequence to cut, mix, degenerate and compound the augmented plasmid; it dose enzyme cutting to the reacted mixture, dispels the parental generation molecule to separate the T carrier. It adopts special biology compositing technique to produce the T carrier.

The invention relates to a method for analyzing a genomic region of an organism, comprising four major parts. The first part involves the isolation of mRNA from a selected organism that is used for the preparation of small single stranded DNA fragments with one adaptor containing an affinity label. These DNA fragments are used in part three. In the second part, genomic DNA from the same or a related organism is isolated. This genomic DNA is fragmented and ligated to adaptor molecules. In the third part, these genomic fragments are hybridized with single stranded DNA fragments from part one, and the hybrids formed in this process are used for synthesis of DNA fragments. These fragments will be used in part four which involves sequencing of these fragments using one of the available high throughput sequencing methods.

The invention relates to the technical field of genetic engineering, and discloses a method for constructing a T vector, in order to directionally clone a resistance gene containing a specific restriction endonuclease recognition sequence at both ends between two multiple cloning sites of the starting vector, Screen positive clones with antibiotics corresponding to the above-mentioned resistance genes to obtain T vector precursors; then digest the obtained T vector precursors with the specific restriction endonuclease, recover large fragments, and obtain T vectors; wherein, the specific restriction endonuclease The endonuclease whose recognition sequence is digested can be a restriction endonuclease whose protruding terminal site can be T, and the resistance gene is different from the resistance gene contained in the starting vector. The method for constructing the T vector in the present invention is simple and efficient, and the quality of the T vector is controlled by using the introduced resistance gene fragment during the preparation process. The T vector obtained by this method not only retains the advantages of the starting vector, but also has the characteristics of stable quality, low self-cyclization rate, and high cloning effect.