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 available from Altogen Biosystems, which includes:

• Astrocyte Transfection Reagent (0.5 ml / 1.5 ml /8.0 ml)
• Transfection Enhancer (0.5 ml), and
• Complex Condenser (0.5 ml)

This is a lipid-based transfection reagent that is optimized to transfect DNA and RNA into astrocyte cells following either a standard or reverse transfection. The protocol for a 24-well plate to transfect astrocyte cells is as follows:

1. Plate 7,500-12,000 Astrocyte cells per well in 0.5 ml of complete growth medium 12-24 hours prior to transfection
2. Wash with 1xPBS and add 0.5 ml of fresh growth medium
3. Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and (referred to a final volume including growth medium)
   • 750 ng DNA (or mRNA), or
   • 30 nM – 50 nM of siRNA (or microRNA)
4. Incubate transfection complexes at RT for 15-30 minutes
5. Optional: Add 2 µl of Complex Condenser. This reagent increases transfection efficiency by reducing the size of transfection complex; however, it may increase cell toxicity
6. Add prepared transfection complexes to 0.5 ml of complete growth medium with Astrocyte cells (from step 2)
7. Incubate cells at 37ºC in a humidified CO₂ incubator
8. Assay for phenotype of target gene expression 48-72 hours after transfection

Optional: Adding Transfection Enhancer reagent can increase transfection efficiency. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection

If the viability of Astrocyte cells being transfected is affected at 16-24 hours post-transfection, changing the growth medium and eliminating redundant exposure of cells to transfectant can decrease the level of cytotoxicity

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.

ASTROCYTES | ASTROCYTE CELL CULTURE PROTOCOL | TRANSFECTION INFORMATION