Method for detecting CHO cell proliferation by using optical analysis and application thereof

A cell proliferation and optical analysis technology, applied in the field of non-invasive optical analysis to detect Chinese hamster ovary cell proliferation, can solve the problems of compound toxicity, reduced accuracy, staining dispersion, etc., and achieve good reproducibility, good repeatability, and operation. convenient effects

Inactive Publication Date: 2015-04-29
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods all have some disadvantages: for example, the BrdU labeling method needs to denature the DNA before it can bind to the antibody, but this destroys the DNA double-strand structure, affects the binding and dyeing of other dyes, and leads to problems such as dyeing dispersion and reduced accuracy; tetrazole The detection methods of salt (MTT, XTT, MTS) belong to the end point detection, the compound is toxic, the sensitivity is insufficient, and the dynamic growth of cells cannot be monitored online in real time; the antigen in the detection metho...

Method used

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  • Method for detecting CHO cell proliferation by using optical analysis and application thereof
  • Method for detecting CHO cell proliferation by using optical analysis and application thereof
  • Method for detecting CHO cell proliferation by using optical analysis and application thereof

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Experimental program
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Effect test

Embodiment 1

[0060] Preparation of pEGFP-N2

[0061] (1) Take a competent cell of Escherichia coli DH5α, put it on ice to thaw for 30 minutes, then take 1 μl of pEGFP-N2 plasmid and add it to the competent cell, and mix well by finger flicking. After 30 min of ice bathing, 42°C water bath for 90 sec, repeat the ice bath for 2 min for transformation. After the transformation is completed, take 0.8ml of SOC medium to suspend the bacteria, and resume culture at 37°C for 1 hour on a shaker at 180rpm.

[0062] (2) Take the recovered bacterial solution, centrifuge and resuspend the pellet, take 10ul and spread it on the resistant LB plate, and culture overnight at 37°C. Pick clones with good growth potential, inoculate them in LB liquid medium containing resistance, and cultivate overnight at 37°C on a shaker at 180rpm.

[0063] (3) Extract the plasmid for sequencing. After the sequencing result is correct, shake the bacteria, use the OMGA plasmid extraction kit to extract the endotoxin-free p...

Embodiment 2

[0069] Liposome lipofectimene3000 transfection and its screening

[0070] (1) The revived CHO cells were inoculated into T-flask culture flasks containing 6ml of growth medium (10% FBS+1% Penicillin-Streptomycin Solution+89% DMEM / F12, hereinafter referred to as 10% FBS medium group), at 37 °C, 5% CO 2 Cultured in a constant temperature cell incubator until the cell density reached 90%. After several generations of culture, inoculate 1×10 5 cells in a 24-well plate at 37°C, 5% CO 2 Culture at constant temperature until the cell growth density reaches 70%.

[0071] (2) Refer to the procedure of lipofectimene3000 kit for transfection operation. Mix lipofectimene3000 reagent and opti-MEM medium uniformly to obtain solution A; mix 1ug pEGFP-N2, opti-MEM medium and P3000 reagent uniformly to obtain solution B. Equal volumes of solutions A and B were mixed in a 1.5ml centrifuge tube, incubated for 5 minutes and then added to a 24-well plate for culture. After 48 hours of transf...

Embodiment 3

[0075] PCR identification of resistant cell clones

[0076] In order to identify whether the target gene GFP is fully integrated into the cell genome, the whole cell genome was extracted using the Takara Genome Extraction Kit.

[0077] (1) Using the genome obtained from lysed cells as a template. The genome template of untransfected CHO-K1 cells was used as a negative control.

[0078] The primer sequences are as follows:

[0079] pEGFP-N2-F2: 5'-CTGGTCGAGCTGGACGGCGACG-3'

[0080] pEGFP-N2-R2: 5'-CACGAACTCCAGCAGGACCATG-3'

[0081] The reaction system is shown in Table 2:

[0082] Table 2 PCR reaction system for cell identification

[0083]

[0084] All experimental operations should be carried out under sterile conditions to prevent cross-contamination.

[0085] (2) Amplify the target fragment according to the PCR program set below.

[0086] PCR program settings:

[0087]

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Abstract

The invention discloses a method for detecting CHO cell proliferation by using optical analysis and application thereof. The method comprises the following steps: (1) converting pEGFP-N2 into Escherichia coli DH5alpha, shaking the bacteria, and extracting endotoxin-free plasmids; (2) inoculating CHO cells into a pore plate, performing transient transfection on the plasmids in the step (1) into the CHO cells by adopting a lipofection transfection method when the cell growth density is 70 percent; (3) screening the transfection cells by adopting geneticin G418, performing resistance screening of over 30 generations at least, thereby obtaining a cell strain which can stably express the GFP; and (4) performing multiplication culture on the cell strain which can stably express the GFP, digesting the cells, performing cell counting by adopting trypan blue, detecting the fluorescent intensity by adopting an enzyme linked immunosorbent assay detector, and judging the CHO cell proliferation conditions based on a linear relation between GFP fluorescent intensity in the cells and the cell number. The method disclosed by the invention has the advantages of operability, detection result accuracy, high repeatability, high sensitivity, non-toxicity and the like.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a non-invasive optical analysis method for detecting the proliferation of Chinese hamster ovary (CHO) cells, which is suitable for rapid screening of medium for promoting the growth of CHO cells and various factors. Background technique [0002] Mammalian cell expression system is the preferred expression system for pharmaceutical proteins: In 1986, the first therapeutic protein-human tissue plasminogen activator was expressed and produced by mammalian cells; according to statistics, from 2006 to 2010, it obtained the Of the 58 biopharmaceuticals approved by the FDA, 32 of them are expressed using mammalian cell lines (Walsh G. Biopharmaceutical benchmarks. Nat Biotechnol 2010; 28:917–924). In many mammalian expression systems, nearly 70% of therapeutic proteins are expressed in CHO cells (Jayapal KP, Wlaschin KF, Hu WS, Yap MG. Recombinant protein therape...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12Q1/06
Inventor 王菊芳钟启马毅刘婷婷王海鹰
Owner SOUTH CHINA UNIV OF TECH
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