Method for transfecting fish or amphibia cell line by using slow virus

A technology of lentivirus transfection and cell line, which is applied in the field of genetic engineering, can solve the problems that the research on lentivirus transfection of fish cell lines and amphibian cell lines has not been reported yet, and achieves strong repeatability, high efficiency, and easy operation. simple effect

Active Publication Date: 2015-11-04
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there have been no reports on lentivirus transfection of fish cell lines and amphibian cell lines

Method used

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  • Method for transfecting fish or amphibia cell line by using slow virus
  • Method for transfecting fish or amphibia cell line by using slow virus
  • Method for transfecting fish or amphibia cell line by using slow virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for transducing fish cell lines with lentivirus, including the following steps:

[0025] In this example, lentivirus packaging and Lipofectamine 2000 promote the lentivirus infection of grass carp kidney cell line (CIK), and the transduction efficiency of lentivirus is detected by observing the expression of green fluorescent protein GFP under a fluorescence microscope.

[0026] 1. Packaging of lentivirus and preparation of virus suspension

[0027] A three-plasmid system was used to prepare the lentivirus, the packaging plasmid pCMVΔR8.2Δvpr, the transfer plasmid DHIV-CTE inserted the green fluorescent protein (GFP) reporter gene, and the outer membrane protein particle pCMV-VSV-G. The three plasmids were all provided by Dr. Yuanan from the University of Hawaii Lu Huizeng (Zeng L, Planelles V, Sui Z, et al. HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1. Journal of Gene Me...

Embodiment 2

[0042] Lentiviral transfection of spathian sturgeon fin ray tissue cell line

[0043] Same as Example 1

[0044] Table 2 Different methods of lentiviral transfection efficiency of S. spathula fin ray tissue cells

[0045]

Embodiment 3

[0047] The steps for transfecting giant salamander muscle tissue cell line with lentivirus include:

[0048] Except for step 2 of embodiment 1, the steps of step 1) are:

[0049] 1) Take a sterile EP tube and add 250μL of serum-free M199 medium, add 10μL of Lipofectamine 2000 and let it stand for 5 minutes, then add the diluted lentivirus suspension (MOI=100) and incubate for 10 minutes, then add it to the cell culture well and place it at 30℃ After culturing in an incubator for 2 hours, transfer to a 26°C incubator for 2 hours, then transfer to a 20°C incubator for 2 hours, aspirate the medium, and add 2 mL of fresh M199 medium containing 10% fetal bovine serum to continue the culture;

[0050] The remaining steps are the same as in Example 1.

[0051] Table 3 Different methods of lentivirus transfection efficiency of giant salamander muscle tissue cells

[0052]

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Abstract

The invention discloses a method for transfecting a fish or amphibia cell line by using slow virus, which comprises the following steps: using Lipofectamine 2000 in the slow-virus target cell infection process; and after the target cell line is infected, carrying out continuous culture, wherein the culture temperature forms a gradient change (30-32 DEG C for 2 hours, 26-28 DEG C for 2 hours and 20-25 DEG C). The method can successfully transfect the fish or amphibia cells by using slow virus, and the transfection efficiency after 72 hours is 85% or above. The method has the advantages of high repetitiveness, high efficiency and low cost, and is suitable to be widely popularized and used in experimental research and fish cell or amphibia cell gene engineering.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for transfecting fish or amphibian cell lines with a lentivirus. Background technique [0002] Lentiviral vector is a gene therapy vector developed based on human immunodeficiency type I virus (HIV-1). Lentivirus-mediated transgenic technology is a hot spot in contemporary gene therapy and transgenic technology research. Lentiviruses can use long terminal repeats (LTR) with autonomous integration functions at both ends of their genome to stably integrate the target gene carried on the chromosome of the host cell, thereby achieving continuous transcription or expression of foreign genes. Lentivirus has the advantages of multiple types of transfected cells, large exogenous target gene fragments (~10Kb), and small immune response. In addition, the virulence gene of the lentivirus is removed and it is split into three or four plasmid systems, namely Trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 马杰曾令兵范玉顶周勇
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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