36results about How to "High homologous recombination efficiency" patented technology

The present invention relates to a method for producing a modified foreign chromosome(s) or a fragment(s) thereof, which comprises the steps of: (a) preparing a microcell comprising a foreign chromosome(s) or a fragment(s) thereof, and transferring said foreign chromosome(s) or a fragment(s) into a cell with high homologous recombination efficiency through its fusion with said microcell; (b) in said cell with high homologous recombination efficiency, inserting a targeting vector by homologous recombination into a desired site of said foreign chromosome(s) or a fragment(s) thereof, and / or a desired site of a chromosome(s) derived from said cell with high homologous recombination efficiency, thereby marking said desired site; and (c) in said cell with high homologous recombination efficiency, causing deletion and / or translocation to occur at the marked site of said foreign chromosome(s) or a fragment(s) thereof.

The invention discloses a method for improving the homologous recombination efficiency of Gluconobacter oxydans, belonging to the field of genetic engineering. The present invention expresses exogenous recombinase in Gluconobacter oxydans, improves the homologous recombination efficiency of the strain, and realizes gene knockout and screening by constructing homologous fragments with screening markers and homologous fragments without screening markers Mark removal. The obtained gluconobacterium oxidans expressing the Red recombinase will play an important role in the construction of vitamin C one-step production bacteria.

The invention discloses a method and application for improving application efficiency of a gene targeting technique in aspergillus terreus, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, by taking Aspergillus terreus as an initial bacterium, knocking off a ku80 gene or an lig4 gene so as to increase the exogenous DNA homologous recombination probability of a strain; secondly, establishing a pyrG gene deletion uracil auxotroph stain, establishing a inheritance conversion system based on a pyrG gene as a screening tag; and finally, cutting off the screening tag by using a Cre / LoxP specific binding site recombinant system, thereby obtaining a uracil auxotroph stain which can be applied to genetic modification again. By adopting the method disclosed by the invention, an efficient aspergillus terreus gene targeting platform can be established, the method has the advantages that high homologous recombination efficiency can be achieved, the bidirectional screening of the conversion system can be achieved, a screening tag cutting method is simple and feasible, the screening tag can be recycled, and the like, and basic support can be provided for efficient genetic modification of aspergillus terreus by using the gene targeting technique.

The invention is applicable to the field of biotechnology, and provides a preparation method of an Escherichia coli capable of automatically degrading nucleic acids during cracking. The method comprises steps of: connecting a pelB signal peptide sequence with an SNase sequence to obtain a first combined sequence; connecting the first sequence with a Km gene sequence to obtain a second combined sequence; substituting a ptsG gene of Escherichia coli with the second combined sequence to obtain Escherichia coli with deletion of ptsG gene; and knocking out the Km gene from the second sequence and in the Escherichia coli with deletion of ptsG gene. The preparation method of Escherichia coli provided by the invention has the advantages of high efficiency of homologous recombination; the SNase expressed by the prepared Escherichia coli can secret into a periplasmic space, and a product expressed by the SNase gene does not influence the expression of other protein; and the method can reduce the accumulation of acetic acid in a fermentation process and promote thallus growth.

The invention relates to a construction method and a kit for a gene detection standard substance based on yeast cells. The yeast cells are creatively used as a genetic background for the first time, to-be-detected target genes such as a human body cell mutation gene are integrated into a yeast cell genome through a homologous recombination mode, and therefore, obtained recombination yeast cells can be used as positive contrast standard substances in genetic detection of target genes.

The invention discloses a method for improving the efficiency of homologous recombination in CHO cells and related products and applications. The invention firstly discovers and proves that knocking out the POLQ gene alone in CHO cells and inhibiting the al-NHEJ repair pathway can efficiently improve the efficiency of CHO cells. Homologous recombination efficiency, the constructed POLQ gene-knockout CHO cells can improve the efficiency of screening stable cell lines when expressing foreign antibodies, and further increase the expression of products, and have a good effect in the preparation of recombinant protein biopharmaceuticals Application prospect.

The invention discloses an efficient CRISPR RNP and donor DNA colocalization-mediated gene insertion or replacement method and its application. The method comprises the following steps: (1) biotin-labeling donor DNA to obtain biotin-labeled donor DNA; (2) fusion protein of Cas9 protein and monovalent streptavidin, sgRNA and biotin-labeled The donor DNA is mixed evenly and left to stand to obtain the CRISPR RNP-donor DNA complex; (3) the CRISPR RNP-donor DNA complex is subjected to nuclear transformation to realize gene insertion or replacement. In the present invention, the fusion protein can be combined with the characteristics of biotin-labeled donor DNA, so that CRISPR RNP and donor DNA co-appear at the target site, so as to realize the precise insertion of the donor DNA at the target site or the gene expression of the target site. Precise replacement, suitable for crop and livestock breeding.