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33results about How to "Knockout Efficient" patented technology

Method for quickly building CRISPR gene editing liver cancer cell strain and cell strain

PendingCN111254164AGood expression efficiencyStable knockoutHydrolasesGenetically modified cellsCas9Liver adenocarcinoma
The invention discloses a method for quickly building a CRISPR gene editing liver cancer cell strain. According to the method disclosed by the invention, a CRISPR/Cas 9 technique is improved, recombinant plasmids better in expression efficiency are constructed, quick monoclone culture is combined, and a stable gene knockout liver cancer cell strain is constructed. Required sgRNA is accurately obtained through primer synthesis, an inserting fragment is synthetized through two-step PCR, a carrier is loaded, and recombinant plasmids for knockout are constructed; after slow viruses are packaged, the packaged slow viruses and liver cancer cells are co-incubated, and sgRNA and Cas9 proteins of equal quantity are transmitted into the liver cancer cells at the same time through slow virus mediating; and liver cancer cells after gene editing are subjected to puromycin resistance screening and monoclone culture, and finally, a gene knock-out positive stable liver cancer cell strain is quickly obtained. According to the method disclosed by the invention, important experimental materials are provided for researching a molecular mechanism of the gene in generation and development of tumors, andreference is provided for in vitro cell modeling of liver cancer diseases.
Owner:DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI

Construction method of CNN3 gene knockout mouse model based on Cre-FloxP system

The invention discloses a construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system, which comprises the following steps: respectively inserting FloxP gene segments at two sides of a second exon of a CNN3 gene, constructing a Cas9 / gRNA target and a homologous recombination template, carrying out microinjection on the homologous recombination template and donor DNA into a fertilized egg of a mouse to obtain a Cnn3-FloxP mouse, and carrying out Cre-FloxP system-based CNN3 gene knockout mouse model; after mating with a female mouse, conducting selfing to obtain a stably inherited homozygote CNN3fl / fl mouse; after the homozygote CNN3fl / fl mouse and a Cre mouse with tissue specific expression are copulated, conducting selfing, and screening out the mouse with the genotype of Cre + / - / CNN3fl / fl through gene recognition. The invention relates to the technical field of gene design, can efficiently construct a mouse with specific tissue or specific cell CNN3 gene knockout, and greatly improves the construction efficiency and the construction success rate of the mouse model with the selective CNN3 knockout function.
Owner:FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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