1. info@altogenlabs.com • 512.433.6177
Provider of Global Contract Research Services Accelerating Preclinical Research, Drug Discovery & Therapeutics
Generation of Stable Cell Lines
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

2. GLP-preclinical and biology CRO services:
Generation of stable cell lines
Other services includes:
A-to-Z RNA interference services
Cell Biology services including in-house/custom cell lines, cell based assay development and antibody production
Molecular biology services including gene synthesis, vector construction, sub cloning and expression
Xenograft mice services
Pharmacology and toxicology studies
Microorganism identification (bacteria ID) services
100% IP rights belong to the client
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

3. Generation of Stable Cell Lines
Altogen Labs offers several stable cell line generation services:
Reporter stable cell lines (luciferase, GFP, RFP, YFP)
Protein overexpressing cell lines
Knockdown (RNAi) cell lines
Tetracycline inducible cell lines
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

4. info@altogenlabs.com • 512.433.6177
Types of Transfection
Transfected genetic material can be expressed in the target cells either transiently or permanently depending on the methods utilized and the experimental questions being investigated.
Transient transfection is used most commonly to analyze the short term impact of altered gene or protein expression. Plasmid DNA (pDNA), messenger RNA (mRNA), short interfering RNA (siRNA), and microRNA (miRNA), are introduced and gene products are expressed in the target cells however the nucleic acids do not integrate into the host cell genome. Therefore, gene product expression is transient and typically results in high expression levels that persist for hours when RNA is transfected, or hours following DNA transfection.
Conversely, stable transfected cell lines are developed in order to analyze the long term impact of altered gene or protein expression. In a subpopulation of transfected cells, the transfected genetic material will integrate into the genome. In order to create stable cell lines, the gene of interest along with a selectable marker is introduced in to the cell. The growth of transfected cells, in the presence of a selecting agent, will enable the subpopulation of cells in which the exogenous genetic material has been incorporated into the genome to persist while the remaining cells undergo selection.
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

5. info@altogenlabs.com • 512.433.6177
Typically, antibiotic resistance or fluorescent reporter gene markers are incorporated into the plasmid DNA construct to facilitate selection process. These selection markers can be co-expressed on the same vector or independently expressed on two separate vectors. The selection process facilitates the selection of the most efficient expressers or silencers of the gene of interest.
For example, Geneticin, also known as G418 sulfate, is commonly used for the selection of mammalian, plant, or yeast cells. Hygromycin B is an aminoglycosidic antibiotic that inhibits protein synthesis by disrupting translocation and promoting mistranslation of the 80S ribosome. Because its mode of action is different from Geneticin, Hygromycin B can be used in dual-selection experiments.
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

6. Steps included in the service
Transfection (10-20µg plasmid/cell line)
Clonal selection in appropriate selection controls (antibiotics or fluorescence tag)
Colony pick up for monoclonal cell line development
Stable cell line generation
Gene cloning (if DNA plasmid not provided by the client)
Expression and stability check (at least 10 passages)
To generate stable cell line
Service completed in 28 days
Validation of construct expression by qRT-PCR and/or Western blot analysis
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •
Delivery of the cell line on dry ice and DNA plasmid (if cloning was requested).

7. Protein Over-expression: Mammalian Stable Cell Line Development
Hybridoma mammalian cell lines hybrid cells produced from the fusion of normal cells with myeloma tumor cells
Characteristics:
Constitutively produce specific antibodies from the primary lymphocytes
Immortalization
Mammalian post-translational modification and protein folding that are crucial in the production of antibodies, recombinant proteins, viral-subunit proteins, and vectors for gene therapy
Non-hybridoma stable cell lines may be produced with integrated plasmids in the genome that also produce gene-specific over-expression of desired proteins.
Importance Gene therapy, new compound screening and therapeutic drug research
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

8. Stable RNAi Cell Line Generation
The mechanism of RNAi is based on the sequence-specific degradation of host mRNA through the cytoplasmic delivery of double-stranded RNA (dsRNA which are typically nucleotides in length) identical to the target sequence.
Potent inhibition is experimentally achieved by the transfection of small interfering RNA (siRNA), which binds RISC complex and causes degradation of target complementary mRNA molecules in the cell. Therefore, successful, potent RNAi experimentation is dependent upon the highly efficient delivery of the siRNA into cells by transfection of stable and functional siRNA molecules.
Generation of RNAi stably-expressing cell lines is very useful in functional analysis of genes, gene target discovery, validation of gene targets, assay development, and screening of drug compounds.
Challenges: very laborious (extensive use of controls such as the efficiency of gene silencing, controlling for off-target effects, and promoter compatibility), expensive, and time-consuming
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

9. Tetracycline inducible cell lines
Analysis of gene function using transient/stable over-expression or knockdown of the gene of interest is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells.
In short, TetR has been modified to either
(1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or
(2) bind to the TO in the absence of tetracycline (termed Tet-on system).
The Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium). Hence the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector system.
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

10. info@altogenlabs.com • 512.433.6177
Tet-OFF System Tet ON system
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •
Gomez-Martinez et al., 2013

11. info@altogenlabs.com • 512.433.6177
Custom-generated clonal stable cell lines
In-house cell lines stably expressing tetracycline repressor protein
Why Altogen Labs?
Unique experience with more than 150 cancer cell lines
Highly efficient gene delivery technologies
We have extensive cell culture experience and expertise, as well as specific optimized protocols for efficient cell line generation
Customizable reporter systems
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •
Cost effective and time managed delivery (Duration- 28 Days)

12. Standard protocol for stable cell lines
HepG2 Standard Transfection Protocol (24-well plate):
Plate 7, ,000 HepG2 cells per well in 0.5ml of complete growth medium 12–24 hours prior to transfection. Wash with 1xPBS and add 0.5 ml of fresh growth medium. Prepare transfection complexes by mixing 40μl of serum free medium, 5.5μl of transfection reagent, and 750 ng DNA (or mRNA), or 30 nM - 50 nM of siRNA (or microRNA) containing gene of interest and G418 antibiotic resistance gene for selection. Incubate transfection complexes at RT for minutes.
Add prepared transfection complexes to 0.5 ml of complete growth medium without antibiotics with HepG2 cells. Incubate cells at 37ºC in a humidified CO2 incubator for 48h. Then exchange the media for growth medium containing 1mg/ml G418. After 14 days of selection, positive cells picked and transferred to 96 well plates. The positive clones were further grown in 400µg/ml G418 and transferred to bigger plates where they reach confluency upon which they were trypsinized and stored in liquid nitrogen for further testing.
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •

13. info@altogenlabs.com • 512.433.6177
Protocol for generation of HEK293S with Tet-On inducible WT opsin (Rho) gene expression
HEK293S cells were transfected with the plasmid pCDNA6-TR, carrying a gene encoding the selectable marker, blasticidin, and a gene coding for the tet operon repressor protein (TetR).
The transfection method used calcium phosphate precipitation. Stably transfected cell lines resistant to blasticidin (5μg/ml) were selected.
The pool of the resulting colonies was then expanded under blasticidin selection and transfected with pACMV-tetO-Rho. Cell lines stably transfected with this plasmid were selected by using Geneticin (2mg/ml), and individual colonies appearing after 14 days were isolated and expanded.
Cell lines were expanded in triplicate to 107 cells in 10-cm-diameter culture dishes. They were supplemented with growth medium alone or with growth medium containing both tetracycline (2μg/ml) and sodium butyrate (5 mM). The cells were incubated for a further 48h (Reeves et al., 2002).
Altogen Labs • 4020 S Industrial Dr • Suite 130 • Austin • TX • • USA •
For an immediate project quotation please contact us at or call Altogen Labs technical support at Please note that experimental details will help us provide an accurate price quote and timeline estimate.