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A method for lentivirus transfection of fish or amphibian cell lines

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: 2018-04-20
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A method for lentivirus transfection of fish or amphibian cell lines
  • A method for lentivirus transfection of fish or amphibian cell lines
  • A method for lentivirus transfection of fish or amphibian cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for lentiviral transduction of fish cell lines, comprising the following steps:

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

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

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

Embodiment 2

[0042] Lentiviral Transfection of Acipenser Acipenser Fin Ray Tissue Cell Line

[0043] Same as Example 1

[0044] Table 2 Efficiency of different methods of lentivirus transfection of sturgeon fin ray tissue cells

[0045]

Embodiment 3

[0047] The lentivirus transfection giant salamander muscle tissue cell line, the steps include:

[0048] Except the step 2 of embodiment 1, the step of the 1st) step is:

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

[0050] All the other steps are the same as in Example 1.

[0051] Table 3 Efficiency of transfection of giant salamander muscle tissue cells with lentivirus by different methods

[0052]

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Abstract

The invention discloses a method for lentivirus transfection of fish or amphibian cell lines, by using Lipofectamine 2000 in lentivirus infection target cells; after the target cell lines are infected, the culture temperature changes in a gradient, specifically 30-32 The method of 2h at ℃, 2h at 26~28℃, and continuous culture at 20~25℃ shows that this method can successfully transfect fish or amphibian cells with lentivirus, and the transfection efficiency after 72h is above 85%. The method of the invention has strong repeatability, high efficiency and low cost, and is suitable for widely popularization and use in experimental research and genetic engineering of fish cells or amphibian cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for transfecting fish or amphibian cell lines with lentivirus. Background technique [0002] Lentiviral vector is a gene therapy vector developed on the basis of human immunodeficiency virus type 1 (HIV-1). Lentivirus-mediated transgenic technology is a hot spot in contemporary gene therapy and transgenic technology research. Lentiviruses can use the long terminal repeats (LTRs) at both ends of the genome to stably integrate the target gene into the chromosome of the host cell, thereby achieving continuous transcription or expression of the exogenous gene. Lentivirus has the advantages of transfecting multiple cell types, carrying large exogenous target gene fragments (~10Kb), and small immune response. And the lentivirus has eliminated the virulence gene, and is split into three or four plasmid systems, namely transfer plasmid (Transfer plasma)...

Claims

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Application Information

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Patent Type & Authority Patents(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