Shanghai Medicilon provides stable cell line services from animal models to cell function detection, has professional stable cell line builders, and can perform cell migration, invasion, apoptosis, cycle, and proliferation detection in the field of cell function detection , Cellular drug screening and other technical services. Stably expressing cell lines refers to the continuous and stable expression of specific genes or interference with the expression of specific genes in cells. The plasmid DNA of the target gene of the stably transformed cell line is integrated into the cell chromosome, so that the cell can stably express the gene for a long time or continue to interfere with the target gene.
Stable cell line service project
1. Gene overexpression stable transfection cell line construction service (gene overexpression polyclonal cell line construction service, gene overexpression monoclonal cell line construction service): the overexpression cell line construction service includes the construction of the target gene overexpression lentiviral vector , Virus packaging, cell transfection and screening. You only need to provide the cDNA plasmid of the target gene and the cell line to be constructed. We will complete the construction and detection of the target gene overexpression cell line according to your requirements, and provide you with high-quality gene overexpression stable cell line.
2. RNAi gene knockdown stable cell line screening service (RNA interference gene knockdown cell line polyclonal construction service, RNA interference gene knockdown cell line monoclonal construction service): Yingmao Shengye’s RNAi gene knockdown cell line construction is based on Lentiviral expression system, generally using pLVshRNA-puro or pLVshRNA-EGFP (2A) puro vector driven by U6 promoter to construct RNA interference stable cell lines, services include interference vector construction, target screening, interference efficiency verification and stable cell screening and clone.
3. CRISPR gene knockout stable cell line construction service: With the emergence of the talen and Cas9/gRNA gene knockout systems, the difficulty of targeted knockout of genes has been greatly reduced. Brand-new technology has gradually knocked out gene knockout from the expensive and time-consuming ZNF system to a simple research tool. Now we can provide gene knockout services for Talen and Cas9/gRNA systems. Including the design of gene knockout targets, the assembly of talen plasmids, the construction of Cas9/gRNA plasmids and the verification of gene knockout efficiency.
Stable transfection cell line service process
a. Vector construction: the foreign gene is constructed on a suitable vector.
b. Determination of cell screening concentration: the concentration of antibiotics in which all cells died in 10-14 days is taken as the screening concentration.
c. Cell seeding: cells are seeded the day before the transfection experiment. The plate density of various cells depends on the growth rate and cell shape of various cells. The cells should reach 60%-80% coverage on the day of transfection.
d. Cell transfection: Transfect the target cells with the constructed vector.
e. Screen the cells with antibiotics.
f. Identification of screening results.
Stable cell line screening method
1. After transfection of plasmids, the monoclonal method is used to screen stable cell lines: the transfection efficiency of most cells is low. For gene overexpression, if the transfection efficiency reaches 40%, gene overexpression is acceptable. However, for gene interference experiments, the transfection efficiency requirements are much higher than gene overexpression, usually plasmid transfection can not meet.
In addition, the plasmid dissociates in the cytoplasm and degrades quickly, which cannot meet the long-term test items; the chance of plasmid transfection integration is extremely low, the construction of stable cell lines is time-consuming and labor-intensive, and the yield is very low. Strain.
2. Viral infection screening stable cell lines: Viral infection methods are more convenient and efficient than plasmid transfection screening monoclonal methods, and are currently the mainstream stable cell line screening methods. Lentiviruses are used to prepare stable transfected cell lines because of their efficient integration, efficient transcription, high expression, wide host range, high infection efficiency, and integration with cell chromosomes without gene rearrangement, making them ideal carriers for stable cell lines.
1. Stability: ensure stable expression of transfected cells within 15 generations.
2. Speedy: The general construction is completed within 1 month.
3. Economic: affordable, high cost performance.
4. Quality assurance: strict identification of foreign genes.