Abstract
Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.
Highlights
IntroductionAs a rule of thumb, gene delivery involves the deliberate modulation of gene expression patterns through the delivery of exogenous genetic material, such as (i) chimeric circular plasmid DNAs (pDNAs), which are hybrid plasmids with an expression cassette containing a specific gene of interest (such as a reporter gene encoding for an traceable protein, e.g., luciferase or a fluorescent protein), (ii) messenger RNA (mRNA), (iii) short regulatory RNAs such as short-interfering RNAs, micro RNAs, and short hairpin RNAs (siRNA, miRNA, and shRNA, respectively), and (iv) antisense oligonucleotides (ASOs) [1,8] into the target site of action (Table 1)
As a rule of thumb, gene delivery involves the deliberate modulation of gene expression patterns through the delivery of exogenous genetic material, such as (i) chimeric circular plasmid DNAs, which are hybrid plasmids with an expression cassette containing a specific gene of interest, (ii) messenger RNA, (iii) short regulatory RNAs such as short-interfering RNAs, micro RNAs, and short hairpin RNAs, and (iv) antisense oligonucleotides (ASOs) [1,8] into the target site of action (Table 1)
Sharply different complexes are generated by adding the plasmid DNAs (pDNAs) solution to a large excess of transfectant solution (for instance at a ratio of ≈1:10 (v/v)) or vice versa, or when DNA solution is added to the PEI equivolume (v/v) (Figure 5a)
Summary
As a rule of thumb, gene delivery involves the deliberate modulation of gene expression patterns through the delivery of exogenous genetic material, such as (i) chimeric circular plasmid DNAs (pDNAs), which are hybrid plasmids with an expression cassette containing a specific gene of interest (such as a reporter gene encoding for an traceable protein, e.g., luciferase or a fluorescent protein), (ii) messenger RNA (mRNA), (iii) short regulatory RNAs such as short-interfering RNAs, micro RNAs, and short hairpin RNAs (siRNA, miRNA, and shRNA, respectively), and (iv) antisense oligonucleotides (ASOs) [1,8] into the target site of action (Table 1). RNA interference mechanisms to shorten mRNA half-life and downregulate translation ASOs [10] Large circular dsDNAs (
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
