Staying on Target: Transient Transfection and Transcription

Transfection is a well-established method for either stably or transiently introducing exogenous nucleic acids into cells. The procedure is frequently employed for siRNA or shRNA experiments, expression of constitutively active or dominant negative constructs, and subcellular localization studies involving proteins fused to readily detectable markers. Expression of the transfected gene of interest results in transcriptional changes in the cell that, when analyzed, give insight into the biological interactions and functions of the corresponding protein. There are indications, however, that transfection itself or sequences in the vector backbone can potentially alter gene expression in a manner unrelated to the function of the gene of interest. To investigate the extent of off-target transcriptional changes induced by transient transfection, L. Jacobsen and S. Calvin of Roche Applied Science (Indianapolis, IN) collaborated with E. Lobenhofer of Amgen, Inc. (Thousand Oaks, CA) to conduct microarray-based gene expression profiling of transiently transfected MCF7 breast cancer cells using four different commercially available transfection reagents at three time points with a control vector or a vector containing secreted alkaline phosphatase (SEAP). Expression profiles were collected from each group of transfected cells and compared to profiles from untransfected cells. Several transcripts were differentially expressed in all four transfected samples, indicating that these changes were common to cells transfected with that particular vector and not specific for the activity of SEAP. Gene Ontology enrichment analysis revealed that the differentially-expressed transcripts were most commonly related to cellular responses to the introduction of foreign DNA (as occurs during viral infection) and the intrinsic cellular immune response. The number of transcripts differentially expressed increased with time following transcription and was also greatly dependent upon the transfection reagent used. From this data, it is clear that the transfection process itself as well as the transfection reagents used impact the transcription of genes, resulting in off-target effects. These effects may confound data analysis and mask the precise activity of the gene of interest, which highlights the need for careful controls in these type of experiments.

(See “Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells”)

Rolling out RNA-primed RCA for Cell-free Cloning

In situations where certain DNA sequences cannot be cloned by the traditional method of insertion into a vector and growth in a biological host (for example, due to their length or repetitive nature), cell-free cloning is an attractive alternative. A recent and innovative technique for cell-free cloning uses multiply primed rolling circle amplification (MPRCA), which can amplify DNA molecules in submicroliter reaction volumes and is flexible in terms of the length or sequence to be amplified. MPRCA is based on rolling circle amplification primed by random DNA primers, which afford the technique its flexibility, but also contribute to its main disadvantage. When the quantity of DNA template is very low, the majority of the amplification product arises from undesired DNA synthesis derived solely from the primers, producing amplicons that are found in control reactions lacking template DNA. H. Takahashi, S. Sugiyama, and their colleagues at the National Agriculture and Food Research Organization (Ibaraki, Japan) investigated whether random RNA primers could effectively promote MPRCA and block synthesis of the unwanted byproducts observed with the random DNA primers. Their hypothesis was based on the fact that the bacteriophage φ29 DNA polymerase used in RCA can use RNA as a primer for DNA synthesis but not as a template. They found that MPRCA using thiophosphate-linked RNA primers can efficiently amplify circular DNA molecules without creating byproducts or requiring submicroliter reaction volumes, allowing for the 10¹²-fold amplification of a single copy of a plasmid. The researchers then demonstrated that MPRCA using RNA primers is suitable for some types of cell-free cloning if a specific dephosphorylation/ligation strategy is first employed. Under these conditions, unwanted ligation products would be linear and easily eliminated by exonuclease treatment. The desired circular products would then be amplified, yielding sufficient product even if the ligation efficiency was low.

(See “Cell-free cloning using multiply primed rolling circle amplification with modified RNA primers”)