RAW264.7 single-cell stable cell line transfected with red fluorescent protein and screening method thereof

A RAW264.7, red fluorescent protein technology, applied in the field of bioengineering, to achieve the effect of good passage stability

Pending Publication Date: 2020-04-14
MAB VENTURE BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, there are few reports on how to construct and screen a RAW264.7 single-cell stable cell line transfected with red fluorescent protein. Therefore, a transfection with good transfection effect and good passage stability has been developed. The RAW264.7 single-cell stable cell line of red fluorescent protein is of great significance to biological research

Method used

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  • RAW264.7 single-cell stable cell line transfected with red fluorescent protein and screening method thereof
  • RAW264.7 single-cell stable cell line transfected with red fluorescent protein and screening method thereof
  • RAW264.7 single-cell stable cell line transfected with red fluorescent protein and screening method thereof

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Experimental program
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Effect test

Embodiment 1

[0055] This embodiment is used to determine the optimal screening dosage of G418, the contents are as follows:

[0056] (1) Take the P5 generation cells of RAW264.7 in the logarithmic growth phase (density: 2×10 5 individual / mL, activity: 96.5%), with 5×10 4 Each well was spread in a 24-well plate with G418-free complete medium (DMEM+10% FBS), placed in CO 2 Incubation in an incubator;

[0057] (2) Prepare 1mg / mL of G418 stock solution, add it into cell culture medium (DMEM+10%FBS) to prepare 7 groups of G418 with different concentrations of 300, 400, 500, 600, 700, 800, and 900μg / mL for screening Culture medium;

[0058] (3) After the 24-well plate was incubated for 24 hours, replace the medium with G418-free complete medium (Control) and 7 groups of G418 screening medium, and continue to change the medium with the corresponding medium every 48 hours for 2 weeks (change solution 3 times), during this process, use a microscope to observe the cell state and growth status at...

Embodiment 2

[0062] This embodiment provides a screening method for RAW264.7 single cell stable cell line transfected with red fluorescent protein, comprising the following steps:

[0063] (1) Prepare the plasmid pDsRED2-C1, confirm the correct sequence of the red fluorescent protein DsRed by sequencing, and prepare the plasmid with a concentration of 377 ng / μL;

[0064] (2) Prepare G418 stock solution 1mg / mL, add it to complete medium (DMEM+10%FBS) to make 500μg / mL and 1000μg / mL screening medium;

[0065] (3) RAW264.7 cells were resuspended in complete medium, and 5×10 4 Each well was spread in a 24-well cell culture plate and cultured for 24 hours; the complete medium was replaced with serum-free basal medium, and serum-free basal medium containing pDsRED2-C1 plasmid and transfection reagent (1 μg plasmid + 2 μl Transfection reagent ( HD TransfectionReagent)+100μL DMEM) for transfection; the transfected cells were placed in the incubator to continue culturing, and replaced with comple...

Embodiment 3

[0070] This embodiment provides a screening method for RAW264.7 single cell stable cell line transfected with red fluorescent protein, comprising the following steps:

[0071] (1) Prepare the plasmid pDsRED2-C1, confirm the correct sequence of the red fluorescent protein DsRed by sequencing, and prepare the plasmid with a concentration of 377 ng / μL;

[0072] (2) Prepare 1 mg / mL of G418 stock solution, add it to complete medium (DMEM+10% FBS) to prepare 400 μg / mL and 800 μg / mL screening medium;

[0073] (3) RAW264.7 cells were resuspended in complete medium, and 5×10 4 Each well was spread in a 24-well cell culture plate and cultured for 22 hours; the complete medium was replaced with serum-free basal medium, and serum-free basal medium containing pDsRED2-C1 plasmid and transfection reagent was added (1 μg plasmid + 2 μl Transfection reagent ( HD TransfectionReagent)+100μL DMEM) for transfection; the transfected cells were placed in the incubator to continue culturing, and r...

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Abstract

The invention relates to an RAW264.7 single-cell stable cell line transfected with red fluorescent protein and a screening method thereof. The screening method includes: (1) adding pDsRED2-C1 plasmidand a transfection reagent into RAW264.7 cells for transfection; (2) applying screening pressure to the cells obtained in step (1); and (3) according to a fluorescence observation result, selecting the positively transfected cells obtained in step (2) for digestion, resuspending, single-cell dilution plate paving, and conducting single-cell stable cell line screening. The method for screening RAW264.7 single-cell stable cell line transfected with red fluorescent protein can acquire a RAW264.7 single-cell cell line with good transfection effect, and the cell line has good passage stability.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a RAW264.7 single-cell stable cell line and a screening method thereof, in particular to a RAW264.7 single-cell stable cell line transfected with red fluorescent protein and a screening method thereof. Background technique [0002] Compared with green fluorescent protein, red fluorescent protein has obvious advantages. First, red fluorescent protein can be used together with GFP to solve some scientific problems that GFP alone cannot solve; secondly, as red fluorescent protein, its excitation and emission wavelengths are longer and can cover GFP. The most important thing is that red fluorescent protein has a low background when imaging in cells, which is more suitable for biological science research. Because of these advantages of red fluorescent protein, it has rapidly been widely used in biological research. [0003] Stable cell line construction is to integr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N5/10C12R1/91
CPCC12N15/85C12N15/65C12N5/0694C12N2800/107C12N2510/00
Inventor 王少雄吕品
Owner MAB VENTURE BIOPHARM CO LTD
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