Method for inducing in-vitro transformation of scallop tissue cells

A technology of tissue cells and scallops, applied in biochemical equipment and methods, botany equipment and methods, cells modified by introducing foreign genetic material, etc., can solve problems such as heavy workload, limited culture time, and easy pollution, etc. To achieve the effect of promoting cell proliferation

Inactive Publication Date: 2011-01-05
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Compared with other tissues, heart tissue is easier to overcome microbial contamination due to the existence of the cardiac pericardium in vitro, but heart cells are terminally differentiated cells that h

Method used

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Embodiment 1

[0020] In the present invention, the forward and reverse primers are firstly designed according to the published SV40LT gene sequence to amplify the 2.1kb SV40LT gene, and the SV40LT gene is connected to the lentiviral expression plasmid under the action of TOPO enzyme to obtain the recombinant lentiviral expression plasmid; Lipofectamine is used to promote transfection TM In 2000, the recombinant lentivirus expression plasmid pLenti6 / V5-D-TOPO / LT ​​was transfected into the packaging cell line 293FT cells. After 24 hours of transfection, observed under the microscope, the cells were in poor condition and rounded in shape compared with before transfection. The 293FT cell culture medium containing the recombinant pantropic lentivirus was collected 72 hours after transfection and stored at -80°C.

[0021] Wash the heart tissue sample of the scallop 3 times with boiled filtered seawater to remove microorganisms on the surface; the special disinfectant solution for the heart tissu...

Embodiment 2

[0024] According to the published GFP gene sequence, the forward and reverse primers were designed to amplify the 700bp GFP gene. Under the action of TOPO enzyme, connect the GFP gene into the lentiviral expression plasmid to obtain the recombinant lentiviral expression plasmid; use the transfection-promoting reagent Lipofectamine TM In 2000, the recombinant lentivirus expression plasmid pLenti6 / V5-D-TOPO / GFP was transfected into the packaging cell line 293FT cells. After 24 hours of transfection, the cells were observed under the microscope. After 48 hours of transfection, the 293FT cell culture medium containing the recombinant pantropic lentivirus was collected and stored at -80°C.

[0025] Wash the heart tissue of the scallop 3 times with boiled filtered seawater to remove microorganisms on the surface; the special disinfectant solution for the heart tissue of the scallop contains 500 units / ml of penicillin, 500 micrograms / ml of streptomycin, 100 units / ml of Boiled and f...

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Abstract

The invention relates to a method for inducing in-vitro transformation of scallop tissue cells, which comprises the following steps of constructing recombinant SV40LT and ientiviruses expression plasmids of a report gene GFP and packaging recombinant pantropic ientiviruses, which can express target genes, by using 293FT cells, and is characterized in that: the scallop tissue cells cultured in vitro, in particular chlamys nobilis heart cells cultured in vitro are infected with the recombinant pantropic ientiviruses, and transformation cells generating fluorescence or having splitting capability are obtained by blasticidin screening. In the invention, an exogenous gene is efficiently transferred to the chlamys nobilis heart cells cultured in vitro by the recombinant pantropic ientiviruses and stably integrated in a genome of the chlamys nobilis heart cells, so that the exogenous gene can be expressed continuously, cell proliferation and division can be promoted, and a basis for further constructing a chlamys nobilis cell system is laid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for inducing transformation of scallop tissue cells in vitro, that is, using a recombinant pantropic lentivirus to mediate the integration of exogenous genes into the scallop cells cultured in vitro. Background technique [0002] Marine bivalves have high economic value and are an important part of my country's mariculture industry. The establishment of an in vitro culture system for marine bivalve cells will build a platform for its basic biology, pathogen infection mechanism, research on new species development, and analysis of functional genes, and will ultimately lay the foundation for the establishment of scallop cell lines. However, most of the current research involves the addition of trophic factors that promote cell division, and the research process is lengthy, with heavy workload and slow progress. [0003] Compared with other tissues, heart tissue is...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N7/01C12N5/10
Inventor 晏萌张志峰孙大鹏林娜王华邵明瑜
Owner OCEAN UNIV OF CHINA
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