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Stable Transfection

Transient and Stable Transfection

The capability to integrate genes into the full DNA sequence of a mammalian cell has a significant impact on biomedical research. 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 transfections are used in most cases to analyze the short term impact of an 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, unlike in stable transfection. Therefore, gene product expression is transient and typically results in high expression levels that persist for 24-72 hours when RNA is transfected, or 48-96 hours following DNA transfection.

Conversely, in order to analyze the long term impact of an altered gene or protein expression, investigators typically utilize stable transfection protocols to develop stable cell lines. In a subpopulation of transfected cells, whether the desired effect is a stable or transient transfection, the transfected genetic material will integrate into the genome. In order to create stable cell lines, investigators will take advantage of this natural occurrence, and introduce the gene of interest along with a selectable marker. Therefore growth of transfected cells, in the presence of a selecting agent, will enable the subpopulation of cells where the exogenous genetic material has been incorporated into the genome to persist while the remaining cells undergo selection. Utilizing this method, investigators are able to develop cells that permanently express specific genes through their incorporation in the cellular genome.

Reporter Gene Assays

Reporter gene assays take advantage of the transfection principle and are used to study gene expression, signal transduction, and other cellular activities. “Reporters” (as these genes commonly referred to) are chosen carefully, depending on their characteristics’ influence and whether these can be efficiently classified and measured. Furthermore, they are regularly used as selectable markers to observe gene expression in studies.

Utilizing reporter genes for research reaps several benefits: they serve as markers, support the accumulation of data from gene expression observation, and other such applications. These characteristics are frequently fluorescent and luminescent properties due to their visibility.

The most common reporter systems to measure transfection efficiency include using a green fluorescent protein (GFP), Luciferase, and β-galactosidase (β-gal) assays. Cells are transiently or stably transfected with a luciferase expression plasmid using a transfection kit at the appropriate reagent-to-DNA ratio. Luciferase expression is measured 24 hours post transfection using at least 3 replicates for standard deviation control.

Stable Transfection Protocol Tips

  • Use high quality reagents and columns to purify your plasmid DNA
  • Use high quality reagents and columns to purify your plasmid DNA
  • Optimize your pDNA concentration and transfection reagent to DNA ratio
  • Use low number cell passage
  • Transfect fluorescent pDNA positive control (lucifirase, GFP, b-gal)
  • Reduce or eliminate antibiotics in the growth media
  • Reduce serum concentration in cell growing medium during transfection
  • Don’t add selection antibiotic too early, at least wait the 72-96 hours required to allow cells to recover after transfection

Service: Development of stable cell lines by Altogen Biosystems:

There are several different options for engineering stable expression of exogenous genes in cultured cells. The choices made can be made easier by consultation with Altogen Biosystems scientists (please contact us at techserv@altogen.com).

Production of protein or expression of  regulatory RNA (i.e. miRNA, shRNA):

  • Effects choice of cells to use
  • Effects choice of expression vector
  • Effects method of screening

Constitutive expression versus inducible expression?

  • Depends on type of downstream experiments
  • Depends on protein properties (i.e. cytotoxicity, anti-mitotic)
  • Inducible systems provide superior negative controls
  • Inducible systems provide unique downstream experimental options
  • Several inducible human cancer cell lines are immediately available

Expression vector: plasmid vs. viral?

  • Viral vectors require more time for vector development, however, less time for drug selection and screening
  • Viral vectors are a good option for hard to transfect cells
  • Use of viral transfected “populations” eliminates the need for identifying single cell clones

What cell line(s) to work with?

Although the choice of cell line to use is usually not flexible a key consideration is the efficiency of stable transfection. If a cell line is known to be difficult to transfect or an uncharacterized cell line is being used then several pilot transfection studies should be performed. A test transfection procedure with multiple transfection methods/reagents can be performed usually within 1 week. Testing includes transfection with a reporter plasmid such as a GFP expression vector and measuring the percentage of transfected cells 48 -72 hours post-transfection. In cases, where very low transfection efficiencies (<10%) are observed consideration of viral expression systems should be considered.

Development of  stable inducible cell line (standard service):

Prior to cell transfection:

  • The expression vector was designed and constructed according to the clients specifications.  In this case a protein encoding sequence was inserted into the inducible expression vector for protein production
  • The target cell line was a readily available inducible cell line
  • The screening procedure was determined to be western blot analysis of the inducible expressed protein.  Antibodies were identified and validated for this purpose

Stable cell transfection and clonal isolation procedure:

Transfect cells with expression vector. Allow recovery of cells, integration of transfected DNA and expression of drug-selection gene (48 hours).

Dilute cells for drug selection and clonal growth. Limited dilution cloning was used to ensure selection of clonal populations (14-21 days).

Transfected cells were two-fold serially diluted and plated into multiple wells of 96 well microtiter plates. The medium was changed to selective medium, replenished every three days and the wells will be visually inspected daily to assess cytotoxic response. Controls: The same serial dilutions were done with non-transfected cells and subjected to both regular medium and drug containing medium. Drug resistance clones were identified at the time non-transfected controls were observe to have 100% cell death. Only serial dilutions resulted in in less than 100% cell growth per well were picked for expansion. Ensuring cell growth in each well was derived from a single cell.

Transient screening assay: An aliquot of transfected cells was analyzed for expression of the transfected gene. Screening for exogenous gene expression is done using endpoint RT-PCR and gel electrophoresis and the results are reported.

Expansion and cryopreservation of drug resistant clones. Drug resistant clones were expanded and two vials of each clone frozen and aliquots of each clone were used for screening.

Screening for inducible protein expression. Each clone was incubated with doxycycline to induce gene expression. Total protein was extracted and subjected to western blot analysis. Two clones observed to have inducible expression were further characterized (as shown below). Cells were induced with doxycycline for 3, 5 and 7 days. Total protein extracts were made and analyzed by western blotting using antibody that specifically recognized the induced protein.  Uninduced cultures were used as the negative control.

stable_cell_transfection

Figure 1. Two drug resistant clones were grown for 7 days under conditions to induce expression of the transfected gene. Cell extracts were made after 0, 3, 5 and 7 days of induction. Western blot analysis was performed to visual the expression of the inducible gene product. The gels were probe with actin antibody to control for any differences in the amount of protein loaded.

Study results and deliverables:

Robust induction of the protein expressed by the transfected expression vector was observed. These two clones were expanded and two aliquots were frozen and delivered on dry ice.

In Vivo Transfection Reagents from Altogen Biosystems

The major challenge in performing RNAi studies in vivo is the effective, directed delivery of functional small RNA molecules into specific tissues. Altogen® In Vivo Transfection Reagents could be conjugated with RNA, DNA, proteins, or small molecules and administered systemically via intravenous (i.v) tail vein injection in order to provide directed gene silencing in specific tissues, including liver, pancreas, kidney, and tumors.

Featured in vivo transfection products from Altogen Biosystems:

In Vivo Transfection Reagents  ||  In Vivo Lipid Transfection Kit  ||  In Vivo Kidney-targeted Transfection Kit

Altogen Custom Services provide specialized biotechnology and pharmaceutical services, including over 50 validated xenograft models, development of stable cell lines, RNA Interference (RNAi) services, assay development, ELISA and Western Blot services, siRNA library screening and transfection services. Generation of stably-expressing cell lines can be very expensive and time-consuming. Altogen Labs offers generation of stable cell line service completed in just 28 days (see service details).