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Original link：Harmony Biology - Construction of Stable Cell Lines
The expression of exogenous genes in cells can be divided into two categories. One is transient expression, where exogenous DNA/RNA does not integrate into the host chromosome. Although it can achieve high levels of expression, it usually only lasts for a few days; One type is stable expression (constructing stable transformants), where exogenous DNA is integrated into the host cell chromosome, allowing the host cell to express the target gene for a long time.
The basic principle of establishing a stable cell line is to clone exogenous DNA into a vector with certain resistance, transfect the vector into the host cell and integrate it into the host chromosome, and screen using the resistance markers contained in the vector. The most commonly used resistance screening markers for eukaryotic expression vectors are neomycin, hygromycin, and puromycin. Cell lines that can stably express the target protein or stably silence specific genes are screened.
Screening method
Screening stable cell lines for lentiviral infection
By utilizing the integrated expression characteristics of lentiviruses to screen stable cell lines, this method overcomes the drawbacks of the long cycle of traditional plasmid cloning methods and can efficiently obtain stable cell lines in a short period of time.
Advantages of using lentivirus to prepare stable strains
(1) Cells have a wide range of applications and can be used for various mammalian cells;
(2) The construction of stable transformation plants has a short time and a long expression duration;
(3) Multiple fluorescent markers and resistance genes are available to meet the requirements of observation experiments.
Stable cell line classification
Mixed clone stable cell lines
Mixed clone cell lines are directly screened for drug resistance after gene transfection, and the screened cells express resistance genes and target genes, but contain various different cell clones. The integration positions and expression levels of target genes vary among different clones.
The screening of mixed clone cell lines is relatively fast and cost-effective. When the transfection efficiency of the cell line to be constructed is high and the expression efficiency of the target gene is high, the expression effect obtained by the monoclonal cell line is not significantly different from that of the mixed clone cell line. In this case, multi clone cell screening is an ideal choice.
Monoclonal stable cell line
Monoclonal cell lines are cell lines obtained by amplifying single cells containing stable integrated exogenous fragments. The expression level of the target gene integration site in each cell is highly consistent, but some phenotypes may be lost. Monoclonal cell line screening has a long cycle and high cost. For cell lines that require subcellular localization experiments, monoclonal cell screening is usually recommended for those that are difficult to infect and have low expression rates.
Service Process
List of constructed stable strains (partial)
Chinese name | Corresponding stable plant | Corresponding cultivation conditions | Cell source |
Astrocytoma of the human brain | U-87 MG-luc-mNeonGreen-Puro | MEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human glioma cells | U251-CMV-EGFP-Puro | DMEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human bladder transitional cell carcinoma cells | T24-EGFP-Puro | RPMI-1640+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human pancreatic cancer cell line | SW 1990-EGFP-Puro | L-15+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human neuroblastoma cells | SK-N-SH-EGFP-Puro | MEM+10%FBS+0.4ug/ml Puromycin | Chinese Academy of Sciences |
Human breast cancer cells | SK-BR-3-EGFP-Puro | DMEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human cervical squamous cell carcinoma cells | SiHa-CMV-EGFP-Puro | DMEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Mouse neuroma cells | Neuro-2a-EGFP-Puro | MEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Mouse neuroma cells | Neuro-2a-CMV-Puro | MEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human breast cancer cells | MDA-MB-468-EGFP-Puro | L-15+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human breast cancer cells | MDA-MB-231-CMV-EGFP-Luc-Puro | L-15+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human breast cancer cells | MCF7-CMV-EGFP-Puro | MEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Mouse lung cancer cells | LLC-CMV-sfGFP-Luc-Puro | DMEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human chronic myeloid leukemia cells | K-562-CMV-EGFP-Puro | RPMI-1640+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human T-cell leukemia cells | Jurkat-CMV-EGFP-Puro | RPMI-1640+10%FBS+0.5ug/ml Puromycin | Chinese Academy of Sciences |
Human endometrial cancer cells | ishikawa-CMV-Puro | RPMI-1640+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human liver cancer cells | HuH-7-CMV-sfGFP-Luc-Puro | DMEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human liver cancer cells | HuH-7-Luc Puro | DMEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
human colon cancer cell | HT-29-EGFP-Puro | McCoy’s 5A+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human liver cancer cells | Hep G2-CMV-EGFP-Luc-Puro | DMEM+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human liver cancer cells | Hep G2-CMV-Puro | DMEM+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human cervical cancer cells | HeLa-EGFP-Puro | DMEM+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human cervical cancer cells | HeLa-RFP-Puro | DMEM+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
human colon cancer cell | HCT 116-CMV-EGFP-Luc-Puro | McCoy’s 5A+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human pharyngeal squamous cell carcinoma cells | FaDu-CMV-EGFP-Puro | MEM+10%FBS+1ug/ml Puromycin | Chinese Academy of Sciences |
Mouse colon cancer cells | CT26.WT-CMV-Luc-Puro | RPMI-1640+10%FBS+8ug/ml Puromycin | Chinese Academy of Sciences |
Mouse colon cancer cells | CT26.WT-CMV-Puro | RPMI-1640+10%FBS+8ug/ml Puromycin | Chinese Academy of Sciences |
Human renal clear cell carcinoma with skin metastasis cells | Caki-1-EGFP-Puro | DMEM+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Hamster kidney fibroblast | BHK-21 [C-13]-CMV-Puro | MEM+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human papillary thyroid carcinoma cells | B-CPAP-CMV-EGFP-Puro | RPMI-1640+10%FBS+1%NEAA+2ug/ml Puromycin | Chinese Academy of Sciences |
Mouse melanoma cells | B16-CMV-Puro | RPMI-1640+10%FBS+1ug/ml Puromycin | Chinese Academy of Sciences |
Mouse melanoma cells | B16-CMV-EGFP-Puro | RPMI-1640+10%FBS+1ug/ml Puromycin | Chinese Academy of Sciences |
Human non-small cell lung cancer cells | A549-CMV-EGFP-Puro | F12K+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Human non-small cell lung cancer cells | A549-luc-mNeonGreen-Puro | F12K+10% FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
Mouse breast cancer cells | 4T1-CMV-Luc-Puro | RPMI-1640+10%FBS+1ug/ml Puromycin | Chinese Academy of Sciences |
Mouse breast cancer cells | 4T1-CMV-EGFP-Puro | RPMI-1640+10%FBS+1ug/ml Puromycin | Chinese Academy of Sciences |
Human gastric cancer cells | MKN-45-CBh-luc2-mNeonGreenA-Puro | RPMI-1640+20%FBS+2ug/ml Puromycin | Concord |
Human lung adenocarcinoma cells | NCI-H1975-CBh-luc2-mNeonGreenA-Puro | RPMI-1640+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
human colon cancer cell | HCT116-CBh-luc2-mNeonGreenA-Puro | McCoy’s 5A+10%FBS+2ug/ml Puromycin | Chinese Academy of Sciences |
