Site specific integration vector capable of removing random integration, construction method thereof and application thereof

Abstract

The invention discloses a site specific integration vector capable of removing random integration, a construction method thereof and an application thereof. The vector comprises a pUC ori, an attB sequence and a multiple cloning site (MCS). The upstream and downstream of the attB sequence is respectively provided with a first loxP sequence and a first rox sequence. A CMV promoter is connected to the upstream of the first loxP sequence. A negative screening gene is connected to the first rox sequence. The downstream of the negative screening gene is connected to an internal ribosome binding site IRES2. The downstream of the IRES2 is a fluorescence labeling gene. The downstream of the fluorescence labeling gene is the MCS. The upstream and downstream of the MCS is respectively provided with a second rox sequence and a second loxP sequence. The direction of the second rox sequence and the direction of the first rox sequence are same, and the direction of the second loxP sequence and the direction of the first loxP sequence are same. The downstream of the MCS is a positive screening element. The application relates to rejection of random integration recombinant cells so as to directly screen false attP site specific integration recombinant cells.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a site-specific integration expression vector, in particular to a site-specific integration vector capable of removing random integration and its construction method and application. Background technique [0002] Research on transgenic animals is now a hot spot in scientific research. However, the existing transgenic animal production technology still has certain drawbacks: the position effect caused by the random insertion of foreign genes, that is, the random insertion of foreign genes into genes related to important life activities of cells causes their abnormal expression, or the Insertion of genes into heterochromatin regions results in silencing of foreign gene expression. [0003] In recent years, Streptomyces phage Integrase is because it can mediate the integration of a transgenic plasmid with an attB site at a pseudo attP site in the mammalian genome, and the integrat...

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Problems solved by technology

[0004] However, There are also different degrees of random integration events in integrase-mediated integration [Thyagarajan, B., Olivares, E.C., Hollis, R.P., Ginsburg, D.S. & Calos, M.P. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Molecular and cellular biology21,3926-3934,], these random integrations greatly reduce The accuracy of site-specific integration mediated by integrase has brought uncertain hidden dangers to the safety of the method; while conventional methods can only be identified by molecular biological means such as PCR, and artificially discarded There are randomly integrated recombinant cells in the integrase-mediated integration reaction, which is time-consuming and labor-intensive

[0005] Somatic cell nuclear transfer technology is an effec

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