CHO Transfection Protocols, Kits, Methods

CHO Background

In 1957, the original CHO cell line was derived by Theodore T. Puck at the Boston Cancer Research Foundation. CHO cells’ popularity since then is due to their rapid growth and high rate of protein production. They are especially useful in research and biotechnology applications that require long duration of stable gene expression and high protein yield. CHO cells can be cultured either as a monolayer or in suspension.

CHO cells are unique in that they do not express epidermal growth factor receptor (EGFR), which makes them convenient for the study of EGFR mutations. Both CHO cells and CHO-K1 cells have had their genomes sequenced, aiding in the elucidation of the inner workings of mammalian cells. There is ongoing work to compare the different genetic mutations of CHO cell lines, evaluating their genotypic and phenotypic qualities for various research applications.

Transfection kits for CHO cell lines can be obtained from commercial entities such as Altogen Biosystems, including CHO Transfection Kit and CHO-K1 Transfection Reagent. Various contract research services using CHO cells are available, including stable cell line generation for protein production, assay development, stable RNAi cell lines, xenograft animal models, etc.

Transfection experiments using the CHO cell line are used to produce recombinant proteins by clinical researchers trying to develop novel drugs and therapies. CHO cells can make excellent vectors in epidemiological research, and with its low mammalian diploid chromosome number of 22, the Chinese hamster’s genome is easy to study as a model for radiation cytogenetics. Additionally, CHO cells are relatively easy to culture, grow quickly, and produce a substantial volume of gene products stably, as compared to other cell lines. This has made them the gold standard cell line within the scientific community, and they are described by some as the mammalian equivalent of E. coli in research.

CHO Cell Transfection Protocol

A pre-optimized CHO cell line transfection kit is available from Altogen Biosystems – CHO Transfection Kit.  The kit is optimized to transfect miRNA, siRNA or DNA plasmid following either a forward or reverse transfection.  The optimized protocol for a 24-well plate to transfect CHO cells is here:

  1. Prepare CHO cell suspension:
    • Trypsinize cells for 3-5 minutes at 37°C
    • Dilute in complete growth medium to 5 x 104 cells/mL
  2. Prepare transfection complexes by mixing 40 µL of serum-free medium, 5.5 µL of transfection reagent, and
    • 750 ng DNA, or
    • 30 nM – 50 nM of siRNA or miRNA
  3. Incubate transfection complexes at RT for 15 – 30 minutes
  4. Plate 20,000 – 30,000 cells per well in 0.5 mL of complete growth medium (from step #1) into culture plate
  5. Add step 3 transfection complexes
  6. Incubate cells at 37ºC in a humidified CO2 incubator
  7. Assay for phenotype or target gene expression 48 – 72 hours after transfection

Figure: GAPDH mRNA levels were quantified using qPCR in CHO cells transfected with siRNAs targeting GAPDH or non-silencing siRNA forty-eight hours after transfection. Data were normalized to18S rRNA as a housekeeping gene and represented as relative to the untreated sample. Data are means ± SD (n=3).


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