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Mammalian Stable Cell Line Development Services

Stable cell lines are widely used in recombinant protein and antibody production, drug screening, gene functional studies and other applications. They can grow continuously over a prolonged period of time and stably carry a genetic modification or express a transgene without significant changes in expression levels.

GeneCopoeia offers state-of-the-art services for establishing stable cell lines for protein overexpression, gene knockdown or genome editing that meet your specific research needs.

 

ORF

Stable cell lines with ORF clones

Overexpression of proteins is critical for many applications, including large-scale antibody production, live cell imaging for localization experiments, and purification for structure-function studies. Traditional overexpression in bacteria often renders eukaryotic proteins insoluble, due to misfolding and/or improper post-translational modification. Therefore, it is highly desirable to be able to express eukaryotic proteins in a physiologically relevant system.

GeneCopoeia offers the generation of stable cell lines that can overexpress virtually any protein of interest in your cell line of choice. Customers can choose to have their own expression cassette integrated into the genome, or from among more than 53,000 human and 32,000 mouse ORFs from GeneCopoeia’s extensive ORF clone collection.

 

Applications

  • Monoclonal antibody production
  • Protein production for biochemical assays
  • Protein production for crystal or NMR structural determination
  • Fusion tagging for live cell imaging
  • Fusion tagging for pulldown/immunoprecipitation
  • Drug target analysis

 

Advantages

  • Largest collection of sequence-verified and expression-ready ORF clones/lentiviral particles reduces turnaround time and ensures high quality.
  • Choice of promoters including inducible promoters for toxic gene expression
  • MAR option for increased open-chromatin site integration
  • Safe harbor knock-in ORF clones available for designated safe site integration and physiological levels of transgene expression
  • DHFR or GS gene amplification available

 

To Order

To request a quote for our stable cell line services, please fill out the inquiry form.

Request a Quote

 

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shRNA

Stable cell lines expressing shRNAs

Down-regulating gene expression is fundamentally important to the study of gene function. In higher eukaryotes, which have been historically difficult to manipulate for making complete genetic knock outs, researchers have used RNA interference (RNAi), which reduces gene expression post-transcriptionally without making permanent changes in the chromosome, irrespective of the chromatin structure or DNA methylation state of the target locus. RNAi causes “knock down”, reducing but not completely eliminating gene expression. Knock down can be advantageous over knock out in applications where complete ablation of gene expression is lethal, or in cases where one wants downregulation to be conditional. In contrast to short interfering RNAs (siRNAs), which knock genes down transiently, short hairpin RNAs (shRNAs) can be integrated into the chromosome to downregulate gene expression stably or conditionally.

GeneCopoeia offers the generation of cell lines stably expressing shRNAs targeting a gene of interest. We will design your shRNAs and integrate them into virtually any cell line you choose.

 

Applications

  • Determination of gene function in cells
  • Expression profiling to identify candidate downstream genes in a network or pathway
  • Drug target validation

 

Advantages

  • Largest collection of sequence-verified and expression-ready shRNA clones/lentiviral particles reduces turnaround time and ensures high quality.
  • Inducible promoters available for toxic gene expression

 

To Order

To request a quote for TALEN or CRISPR based stable cell line services, please fill out the inquiry form.

Request a Quote

 

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CRISPR

Stable cell lines constructed with CRISPR

Genome editing-the ability to make specific changes at targeted genomic sites in complex organisms-is of fundamental importance in biology and medicine. Over the past several years, researchers have developed chimeric DNA binding proteins complexed with nucleases to stimulate double strand breaks at defined genomic loci, allowing the ability to insert, delete, and replace genetic information at will. Recently, the Clustered, Regularly Interspersed, Short Palindromic Repeats (CRISPR) systems have gained favor as genome editing tools.

Along with CRISPR-based plasmid design and construction services, GeneCopoeia offers the generation of stable cell lines carrying specific, targeted modifications created with either CRISPR. In addition, genes can be stably activated or repressed using CRISPR activation (CRISPRa) or CRISPR inhibition (CRISPRi), respectively. We will design the CRISPR target site, and, if needed, donor constructs used for homologous recombination-mediated applications, transfect your cell line of choice, and isolate single or double allele-modified clones. Our stable cell line service can also be used with our human and mouse Safe Harbor Integration system and knock in ORF clones.

 

Applications

  • Gene knock out
  • Introduction of point mutations or defined insertions/deletions
  • Correction of disease mutations back to wild type
  • Transgene knock in at a customer-specified locus or at human and mouse Safe Harbor sites
  • Gene tagging
  • Promoter or gene replacement
  • Gene activation
  • Gene repression

 

Advantages

  • GeneCopoeia is an expert in CRISPR technology and a full service provider for genome editing projects.

 

To Order

To request a quote for CRISPR-based stable cell line services, please fill out the inquiry form.

Request a Quote

 

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Cell Immortalization

Primary cells undergo a finite number of population doublings, and reach a state called replicative senescence after a certain number of divisions that limits the proliferation of aged or damaged cells. In order to make primary cell lines continue to grow and divide indefinitely in vitro, an immortalization process is applied to primary cells to significantly prolong their lifespan. Immortalized cells are important models for studies in cell biology, cellular metabolism and differentiation; and can be used for research in cancer biology, immunology, neurology, hematology and more.

Cell Immortalization Strategies

SV40T – The most reliable and simplest way to immortalize many types of primary cells. The viral gene SV40 large T antigen is introduced into primary cells to override the cell cycle by inactivating tumor suppressor genes that induce cellular senescence.

hTERT – One of the most popular approaches to cell immortalization. The telomerase reverse transcriptase (TERT) protein is inactive in most somatic cells, causing the length of telomeres to shorten with age, leading to senescence. Expression of hTERT prevents the telomere from being truncated during cell division. Thus cells are able to avoid replicative senescence. 

 

To Order

To request a quote for our primary cell immortalization services, please contact us at inquiry@genecopoeia.com.

 

Cell Line Characterization

Cell Line Characterization & Authentication

R&D involving the use of cell lines requires a good understanding of the purity, species of origin, chromosomal and genetic characteristics of the cell lines used. Cultured cell lines in the lab are susceptible to contamination by microbes, mycoplasma and other cells, therby demanding periodic monitoring for characteristics to authenticate the cell line identity. GeneCopoeia offers cell line characterization reagent kits and services that involve testing for a combination of important genetic markers, including chromosomes, DNA fingerprints, alternative splicing and genes of your interest to analyze genetic variation, exploring transcriptome profiles, and assure culture sterility.

Please contact GeneCopoeia at 1-866-360-9531 or inquiry@genecopoeia.com for the cell line services.

 

Analyze Genetic Variation

Fluorescence in situ hybridization (FISH)

For chromosome enumeration, as well as detection of disease-related genetic mutations, amplification and rearrangements resulting in large-scale changes in the physical arrangement of genes on chromosomes. More details…

 

Chromosome enumeration FISH probes can be used to determine chromosome copy number in diagnosis and prognosis of cancers and other diseases.

Disease gene FISH probes can be used to detect oncogene deletion, amplification and rearrangement in cancer cellls.

ddPCR™

Droplet Digital™ PCR assesses absolute quantification of genomic DNA for CNV analysis and cDNA for gene expression analysis; ddPCR can also be used for variant allele frequency (VAF) analysis and verification of NGS results.

STR analysis

Cross-contamination among cultured cell lines is a prevalent and persistent  problem. Short Tandom Repeat (STR) DNA profiling identifies polymorphisms among STR markers; it is a relatively easy, low cost and reliable method for the detection of cellular cross-contamination to reveal true cell line identity.

MSI analysis

MSI is failure to correct the errors that spontaneously occur during DNA replication, resulting in the accumulation of chromosomal mutations at certain repetitive DNA motifs as microsatellites. MSI testing is performed to analyze colon and other tumor tissue samples for features suggestive of Lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC).

 

Exploring Transcriptome Profiles

RT-qPCR

For the quantification of alternative splicing variants and gene expression profiling. Custom qPCR primers designed for the quantification of mRNA and detection of exon skipping, intron retention, alternative 5' or 3' splicing and other splicing variants to identify and quantify different splicing variants.

 

Assuring Culture Sterility

Mycoplasma detection

Mycoplasma contamination of cell cultures can easily go undetected and adversely affect cell physiology and metabolism. Therefore, routine testing is recommended to ensure the purity of cultured cells. GeneCopoeia offers Mycoplasma Detection Services as a preliminary testing step prior to all mamallian stable cell line services using our own highly sensitive mycoplasma detection kit.

 

 

FAQ

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.