Cell Line Generation

ALSTEM offers one-stop shop services for establishing stable cell lines with customized genetic modifications at unbeatable prices with rapid turnaround.

Highlighted Features

Service Scope

Luciferase/Nanoluciferase/GFP/RFP pre-labeled stable cell line

HiBiT tagged stable cell line

Target gene overexpression stable cell line

TET (on/off) inducible cell line

Customized stable cell line generation

Note: Detailed information regarding target gene and type of modification required (please fill out a service requisition form)

Customer Provides

Customers' 2 vials of typical storage size Desired Cells

Indication of appropriate growth medium and conditions

Coated 6-well plates and T25 flasks if the cells require specially treated culture vessels

Note: For your own DNA, please provide between 10-100 µg of DNA. We also offer plasmid amplification and custom vector construction for an additional fee.

Deliverables

Two vials (>1 x 10⁶ cells/vial) of pooled cells and/or single clone cells

Milestone reports and detailed final report

Note: If your cells require media other than DMEM or RPMI, please provide 1–2 L of the appropriate medium along with any necessary growth factors.

Our stable cell line generation service workflow.

Timeline & Milestones

Services	Description	Deliverables	Timeline
Construct generation (optional)	Subclone gene of interest into lentiviral or expression vector.	Sequence confirmation Construct summary	1 week
Virus packaging / DNA preparation	Packaging high titer lentivirus Titer evaluation via qPCR	Titer report	1 week
Cell engineering	Stable transfection and single clone screening Detect the expression level by ELISA, WB, FACS and etc	Report QC data	4–8 weeks

Case Studies

1. Luciferase and tdTomato dual-labeled HeLa stable cell line

Firefly luciferase is commonly used as a genetic reporter to assess the transcriptional activity of a construct with luciferase gene under the promoter of interest. We first tested the function of two different promoters, SFFV and CMV, which drive the expression of luciferase, on five different cell lines. Although luciferase activity varies in different cell lines, the SFFV promoter showed much stronger luciferase expressing than that of CMV promoter in each cell line (Figure. 1). Here, Luciferase and tdTomato dual-reporter HeLa cell line is derived from HeLa cells by transduction of ALSTEM's pLenti-SFFV-Luciferase-PGK-tdTomator-T2A-PURO Lentiviral Reporter (cat# LV452) lentivirus. The cell line stably expresses firefly luciferase at high level since it is driven by SFFV promoter. It also expresses a red fluorescent protein marker (tdTomato) and puromycin resistance gene, which are driven by PGK promoter (Figure 2). Luciferase/tdTomato dual-reporter HeLa cells can be a very useful cell line for non-invasive visualization in both in vitro and in vivo experiments.

Figure 1. Comparison of the efficiency of SFFV and CMV promoters to drive the luciferase expression. Each cell line was infected using in house packaged SFFV/CMV_luciferase lentivirus at same MOI (~2-3). The results suggested that the luciferase activity varies in different cell line, while the SFFV promoter shows much stronger luciferase expression than that of CMV promoter in all cell lines.

Figure 2. Panel A: RFP Fluorescence. Dual-labeled HeLa cell line expressing tdTomato at 90% cell confluence. The image was taken using a Nikon fluorescent microscope. Panel B: Luciferase activity. Serial dilutions of dual-labeled HeLa cells were plated into a 96-well plate. The luciferase activity was tested 6 hrs later. Panel C: Dose-response luciferase activity analyses in HeLa cell line after Staurospine treatment. Shown is luciferase activity change of HeLa cells after incubation with increasing concentrations (μM) of Staurospine for 24h. Panel D: Kinetic cytotoxicity assay. Shown is HeLa cell cytotoxicity onset in response to Staurospine dosing (EC50= ~0.1 uM). Data are expressed as mean /- SD from duplicates of three independent experiments.

Vector Information

Figure 3. The lentiviral reporter vector contains luciferase gene driven by SFFV promoter, tdTomato and puromycin resistance gene by PGK promoter.

Key Features:

Excellent signal/background ratio
Stable luciferase and tdTomato expression
Ideal for in vivo bioluminescence imaging and cell-based assays for cancer research.

2. Target gene overexpression stable cell line

Recombinant stable cell lines are one of the widely used tools in drug discovery, toxicity testing, and basic research. Long-term stable expression of a gene of interest (GOI) is usually achieved by transfection or viral transduction of a vector containing the expression cassette of the GOI together with a selection marker (antibiotics or fluorescent proteins). In contrast to transient expression, stable cell line offers reproducible results for assay studies. Here, we packaged lentivirus and transduced a mouse cell line. The expression of transgenes was confirmed by FACS analysis Figure 4.

Figure 4. FACS analysis of a mouse cell line transduced with lentivirus. Assay was run on the transduced and parental cells. The left panel shows transduced cells stained with antibody against target protein verse parental cells, while the right panel transduced cells stained with another antibody against target protein verse parental cells. Both antibodies gave at least one log intensity positive shift (PE channel).

Cell Line Generation

Stable Cell Line Generation

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