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Stable Cell Line Services

Answer: There are two types of related services we currently offer in various cell lines. One consists of the generation of mammalian stable cell lines for protein expression, gene knockdown, CRISPR- or TALEN-mediated genome modifications, and primary cell immortalization. The second type is cell-based validation, in which we offer validation of ORF and cDNA expression levels, shRNA knockdown efficiency, CRISPR sgRNA validation, interaction between miRNAs and their miRNA targets, and promoter activity.

Answer: What is included in the cell-based service depends on the type of service you are interested in, as follows:

1. ORF expression. We will isolate either stable pools or single cell clones carrying stable integration of the ORF of your gene of interest. By default, we validate expression of the ORF by qPCR, although other validation methods (e.g. western, FACS, ELISA, etc.) are also available.

2. Knockdown by shRNA. We will isolate either stable pools or single cell clones carrying stable integration of the shRNA-expressing construct. By default, we validate downregulation of expression of the target gene by qPCR, although other validation methods (e.g. western, FACS, ELISA, etc.) are also available.

3. CRISPR genome editing. We offer virually any CRISPR-mediated applicaiton, including knockout, gene knock-in, mutagenesis, fusion tagging, gene activation, and gene repression. By default, we validate by sequencing the chromosome to detect the presence of the desired modification. Other validation methods (e.g. western, FACS, ELISA, etc.) are also available.

4. Primary cell immortalization. We typically offer immortalization of primary cells by infecting them with lentiviruses expressing either SV40 large T antigen, hTERT, or a combination of both. Using both SV40 large T antigen and hTERT together usually results in longer-lasting immortalization. We can also immortalize using other oncogenes, such as c-Myc, if desired. By default, we will check for expression of the immortalization gene by qPCR and to see if the cells are capable of fifteen or more generations (doublings). Other validation methods (e.g. western, FACS, ELISA, etc.) are also available.

5. Cell-based validation. We will provide a complete validation report containing the original test results and further analysis. The following methods are provided by default. Customers may also request other methods for validating or screening clones:

 

  • ORF expression validation: qRT-PCR, western blot and fluorescence microscopy (only for vectors with a fluorescence reporter gene)
  • shRNA: qRT-PCR
  • miRNA target validation: Luciferase reporter assay and fluorescence microscopy (only for vectors with a fluorescence reporter gene)
  • Promoter activity validation: Luciferase reporter assay or fluorescence microscopy (only for vectors with a fluorescence reporter gene)

 

Answer: For shRNA expression clones provided by GeneCopoeia, we guarantee that at least one in a set of three (3) shRNA constructs generates a knockdown efficiency of at least 70%. Customer-designed shRNAs do not carry this guarantee. Be aware that the knockdown efficiency of shRNAs depends on many factors, such as variability among different cell lines. We can identify the shRNA with the best knockdown performance in customer designated cell lines, and provide a detailed validation report. However, other shRNA constructs may work better in other cell lines. Therefore, if another cell line is needed in a future experiment, it is better for customers to also have shRNAs validated in that cell line for pre-screening.
Answer: We offer services in many cell lines such as HEK293, CHO, HeLa and NIH 3T3. However, if your desired cell line is not in this group, you may need to provide your cell line to us, or we might need to purchase it form a third party. After we perform quality control analysis, the project can be started. Please note that cell lines provided to us must be mycoplasma-free. We perform mycoplasma testing on all cells submitted to us or purchased from a third party..
Answer: This depends on what kind of vector you choose for your promoter clone. For example, our PG04 vector carries Gaussia Luciferance (Gluc) and Secreted Alkaline Phosphatase (SeAP), our PG02 vector carries Gluc, our PF02 vector carries GFP, and our PM02 vector is marked with mCherry. For the PG04 and PG02 vectors, we use the GeneCopoeia Secrete-Pair™ Dual Luminecence Assay Kit and the GeneCopoeia Secrete-Pair™ Gaussia Luciferace Assay Kit. We use fluorescence microscopy for the PF02 and PM02 vectors. We can validate your target promoter in a specific cell line for you as part of a complete service to support your research needs, sparing you much time and work.
Answer: Many plasmids have selectable markers like antibiotic resistance genes. We can introduce the genes of interest by plasmid transfection. Alternatively, if the desired cell line does not transfect well, we can use lentiviral transduction. Once the target gene is stably integrated into the host cell’s genome, antibiotics can be added to the growth medium to select for stable cell lines expressing the inserted gene. We can also use FACS to sort cells expressing fluorescent markers such as eGFP. Expression levels can be assayed by methods such as qPCR, western blot and ELISA.