RNA interference (RNAi) Imaging.

Molecular imaging of gene function by means of RNA interference (RNAi) is powerful, real time  methodology.

RNA interference (RNAi) is a naturally occurring phenomenon and refers to a mechanism of post-transcriptional gene silencing by which double-strand RNAs (e.g. siRNA, shRNA, and miRNA) inhibit gene expression. The dsRNA nucleotides bind to the homologous nucleotide sequence of the targeted messenger RNA, causing mRNA degradation and thus preventing protein production. Targeted RNAi gene knockdown is a highly efficient technique in comparison to traditional gene knockout technology, facilitating the study of functional genomics in vitro in cell lines as well as in vivo in live animals.

Vector co-expression of RNAi with green fluorescent protein (GFP) or firefly luciferase (Luc2) serves as a marker for infectivity and allows tracking and imaging of the RNAi knockdown cells in real time. GFP is an excellent marker for microscopy and high throughput in vitro screening of cell lines. Switching to the firefly luciferase Luc2 (*) reporter for in vivo applications will provide the highest in vivo sensitivity.

Initially, RNAi treatment was accomplished through delivery of siRNA oligonucleotides. Next, plasmid expression was made possible with the generation of precursor short hairpin (shRNA) plasmids. More recently, it was discovered that the use of convergent promoters can avoid hairpin loop structure design. Thus cloned siRNA is significantly less likely to form secondary structures, allowing easier plasmid propagation and sequencing. The convergent promoter design also enables longer siRNA designs of 27- 29bp oligos, which have been shown to be more efficient than traditional 21mers in specific gene knockdown.

Convergent promoters are double-promoters that are modified RNA polymerase III promoters (H1 and U6) that flank the siRNA template. After transcription, the resulting double stranded siRNA product has the same structure as “natural” double-stranded siRNA and does not require the “dicer” processing step—as is the case with short-hairpin RNA. Additionally, hairpin structures are unstable during bacterial replication of the vector. Therefore, hairpin-less vectors prove enhanced stability. Furthermore, the siRNA constructs are shorter, less expensive and easier to clone.

Lentivirus technology is an ideal vehicle for stable and safe RNAi delivery to target cells. Due to its high transduction efficiency, lentiviral vector based siRNA is considered to be the most efficient delivery method available. Lentiviral siRNA facilitates efficient and stable gene knockdown in both dividing and non-dividing cells, even including transfection-resistant cells such as primary cells.

In conclusion, in order to decipher the function of all genes in the (human) genome, lentiviral siRNA is a highly effective technology, facilitating efficient design and delivery from single siRNA constructs, to the construction of complex siRNA libraries.

For custom siRNA plasmid constructs that co-express GFP or Luc2 and can be used for plasmid transfection or lentiviral packaging, please visit http://www.invivoimagingsolutions.com

(*) Firefly luciferase Luc2 is codon optimized for mammalian cytoplasmic expression, as opposed to weak peroxisomal expression of native luciferases. In mammalian cells, firefly luciferase shifts its emission maximum to 608nm – this is advantageous since tissue is transparent to light >600nm. Bioluminescent luciferase offers higher sensitivity in vivo in comparison with fluorescent proteins, because of a favorable signal to noise ratio – the excitation light used with fluorescence imaging generates significant, non-specific tissue autofluorescence, inexistent with bioluminescence. 

Reference:

Characterization of phosphoglycerate kinase-1 expression of stromal cells derived from tumor microenvironment in prostate cancer progression. Wang et al., Cancer Res. 70(2)471-80, 2010.