Transfection Information

Astrocyte Transfection

Astrocyte transfection is the introduction of foreign molecules and genetic material into cultured mammalian cells.

Transfections can be transient or stable, and the process is utilized in biological research to study gene function and modeling the effect of gene therapy on cellular expression. In transient transfections, the short term impact of altered gene function is examined, with high expression of the transfected molecule for 6 to 72 hours. Stable transfection requires that target genes be integrated into the genome of a cell in such a way that expression persists through the life of the cell line and during cell selection at each round of subculturing.

pDNA and mRNA can be introduced via transfection, as well as siRNA and miRNA, which disrupt the development of gene products. This method of post-transcriptional gene silencing has a huge potential in terms of drug therapies that target genetic disease at the gene itself, rather than relying on symptomatic or higher level treatment.

There are a great variety of carrier molecules on the market today which enable scientists to introduce genetic material into cancer or primary cells through non-viral means. This can be done using derived primary cells, but also in vivo. However, all cells are different and require different transfection reagents, carrier molecules, transfection protocols and reagents in order to successfully express a vector gene of interest. For links to resources for transfection and biological services, products and reagents see below.

Astrocyte Transfection Efficiency

Transfection is a major laboratory method to integrate protein, RNA and DNA molecules into cultured cells.  Transfection efficiency is a phrase that is used to sum up the success of an experiment that contains many variables.  A successful transfection depends upon the optimization of these factors:

  • cell type
  • cell density
  • type of transfection reagent
  • transfection volume
  • concentration of test article
  • ratio of test article to transfection reagent
  • media components used (i.e. medium, serum, antibiotics)

Transient gene knockdown using siRNAs can show results as early as 4 hours post-transfection, with maximal effects at 24-48 hours and compete loss of transient effect at 96 hours.  However, the analysis of transfected plasmid DNA needs to be altered to allow DNA uptake, expression of the plasmid and efficacy of the exogenous target gene.  Timing of these cellular events is often overlooked.

There are several pre-optimized transfection reagents commercially available from Altogen Biosystems (https://altogen.com/products-index/) for such cell lines as: MEF, A549, HepG2, Fibroblast, VERO and many others.

Astrocyte Transfection Protocol

A pre-optimized astrocyte transfection kit is commercially available from Altogen Biosystems – Astrocyte Transfection Kit.  The kit is a lipid-based transfection reagent that is optimized to transfect cells following either a standard or reverse transfection.  Here is the protocol to transfect astrocyte cells in a 24-well plate:

  1. Prepare astrocyte cell suspension:
    • Use trypsin for 3-5 minutes at 37°C to detach cells
    • Dilute resultant suspension 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/mRNA, OR 30 nM – 50 nM of siRNA/microRNA
  3. Incubate complexes at RT for 15 to 30 min
  4. Optional 2 µL addition of Complex Condenser: This reagent reduces the size of the complexes; thus, increasing transfection efficiency.  However, it may increase cell toxicity.
  5. Plate 15,000 to 25,000 cells per well in a total of 0.5 mL
  6. To the wells, add the prepared transfection complexes (from step 3 or 4)
  7. Incubate cells at 37ºC in a humidified CO2 incubator
  8. Perform analysis 48 – 72 hours after transfection

Figure: siRNAs targeting LMNA or a non-silencing control siRNA were transfected in astrocyte cells. 48 hours post-transfection, cells were analyzed by qPCR for gene expression levels and normalized to 18S rRNA levels for input variances. All values are determined as relative to untreated samples. (n=3)

Links

Purchase Astrocyte Transfection Reagent

Cell Transfection Products

Transfection. Cells and Molecular Biology Research Methods, Protocols, and Lab Techniques

Biology Transfection Forum

Astrocyte Research Articles and References

Laboratory model of blood-brain barrier: In an effort to make an accurate in vitro model of the blood-brain barrier, this study co-cultured astrocytes and endothelial cells on opposite sides of a custom ultra-thin, highly porous silicon nitride membrane. It was thought that a custom membrane that was thinner and more porous would be better than commercial solutions. Although both types of cells cultured successfully, the custom membrane solution was determined to be no better than commercial membranes.  Article: Astrocytes and the important role in the future research of brain.