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Construction of a genome-wide high-throughput cloning vector

A whole genome, cloning vector technology, applied in the field of biomedicine, can solve the problems of low cell fusion rate, poor screening method, and difficulty in culturing fused cells.

Active Publication Date: 2021-04-23
翁炳焕
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Problems solved by technology

[0010] In order to solve the problems of low cell fusion rate, poor screening method, difficult cultivation of fused cells, and difficulty in the industrial amplification of the whole genome, the inventors proposed the present invention

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  • Construction of a genome-wide high-throughput cloning vector
  • Construction of a genome-wide high-throughput cloning vector
  • Construction of a genome-wide high-throughput cloning vector

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Embodiment Construction

[0026] Combine below figure 1 , figure 2 , image 3 and Figure 4 , the embodiments of the present invention are described in detail.

[0027] 1. Construction of retroviral recombinant vectors pLPCX-hTERT and pLXN-SV40LT

[0028] Subcloned hTERT from PIRES2-EGFP-hTERT to the EcoRI site of pLPCX to construct the retroviral recombinant vector pLPCX-hTERT; subcloned SV40LT from pMFGSV40tsLT to the EcoRI / BamHI site of pLXN to construct the retroviral recombinant vector pLXN -SV40LT.

[0029] Reagents: pLPCX, pLXSN retroviral vectors are products of Clontech, USA; PIRES2-EGFP-hTERT or pCIneo-hTERT or, SV40DNA (strain 776) are products of Invitrogen, USA; PCR amplification kit and calcium phosphate precipitation transfection kit Purchased from Invitrogen Company in the United States; E.coliDH5-alpha Escherichia coli was preserved as the unit; Endotoxin-free plasmid purification kit was purchased from QIGEN Company in Germany; EcoR I, Not I, BamH I, and endonucleases were from ...

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Abstract

A construction of a genome-wide high-throughput cloning vector used in the field of biomedicine, characterized in that a retroviral vector containing hTERT and SV40LT is constructed, transfected into PT67 packaging cells, and the plasmid-packaged PT67 cells are obtained by joint screening with G418 and PM , extract its culture medium, transfect sp2 / 0 cells, and then use G418 and PM to jointly screen to obtain highly immortalized plasmid-packed sp2 / 0 cells, which can be screened by HAT, G418 and PM after implanting the exogenous whole genome To obtain a highly immortalized hybrid strain that can freeze and amplify the whole genome, it is used for the industrial preparation of the whole genome of rare diseases and the construction of the immortal gene bank.

Description

technical field [0001] The invention relates to the construction of a whole-genome high-throughput cloning vector in the field of biomedicine, which is mainly used for the collection and industrial preparation of the whole genome of rare diseases, and provides renewable whole-genome resources for clinical, scientific research, pharmaceutical and diagnosis. Background technique [0002] In order to better carry out research on pathogenesis, clinical treatment and experimental diagnosis, more and more domestic and foreign institutions have focused on the collection of various case samples and the construction of sample banks. [0003] Various types of cell banks have been established in the United States, the United Kingdom, Japan and China, such as the American Standard Cell Bank (ATCC), the Human Genetic Mutation Cell Bank (HGMR), and the Cell Aging Cell Bank (CAR); the British Embryonic Stem Cell Bank Japanese deciduous tooth stem cell research bank; China's important medic...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/65C12N5/10
Inventor 翁炳焕黄荷凤马端
Owner 翁炳焕
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