Michael Court
May 2004
Transient and stable gene expression using in HEK293 cells
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Plasmid preparation
a. Plasmids for cell transfection must be very pure
i. Maximizes transfection efficiency
ii. Minimizes cell death from endotoxin
iii. Do not use Qiagen miniprep or equivalent
b. Prepare 250 mL bacteria culture
c. Purify with Qiagen Endofree Maxi Prep Kit
i. Could also do 2X cesium chloride purification instead
Linearization of expression plasmid
a. Recommended for stable cell lines
b. Choose restriction site in non-essential location
i. Not in or near genes that are to be expressed (your inserted gene or the
eukaryotic selection gene – e.g. Neo)
c. Increases chance that integration doesn’t occur in your inserted gene
i. This is most common reason for growth of resistant cells that don’t express your
inserted gene
Transfect cells
a. Recommend using individual 12.5 sq.cm flasks (unless too many transfections)
b. Use either Lipofectamine 2000 (Invitrogen) or Fugene 6 (Boehringer-Manheim)
c. Include positive control to monitor transfection efficiency
i. e.g. Green fluorescent protein (GFP) tagged CAT
ii. Either as separate transfection, cotransfection, or GFP fusion
Evaluate transfection efficiency
a. After about 2 days
b. Count percentage of GFP positive cells
i. Commonly 10 – 80%
c. Could also quantitate mRNA expression of your inserted gene by RT-QPCR
i. Requires harvesting at least 10% of cells (return rest to flask
Harvest cells for your experiment
a. Can return 10% of cells to flask for stable cell line (next)
Generate stable cell line
a. 48 hours after transfection add media with selection agent
i. 800 ug / mL Geneticin (G418)
b. Allow up to 2 weeks for selection and growth of resistant clones
i. Change media when old media starts to turn yellow
c. Transfer visible clonal colonies to 6 well plate
i. Collect at least 6 – 12 colonies
ii. Change to media with antibiotics (Pen/Strep and half strength selectyion
antibiotic [400 ug/mL Geneticin]) immediately before picking colonies
iii. Do under sterile conditions as possible
d. Allow to grow to about 80% confluency
i. Maintain antibiotics
e. Harvest about 90% of cells and evaluate for expression of your inserted gene
i. RT-QPCR (best for gene sequence variants)
Michael Court
ii. Activity if enzyme
iii. Western blot if have antibody
f. Transfer best 2 – 3 clones to T-75 for scale up production
May 2004