RNA interference (RNAi) is an endogenous system that regulates gene expression. Since its discovery (Fire et al, 1998
) it has been exploited to silence specific genes and has become an important experimental method utilized in cellular and in vivo
studies. RNAi based therapies are in development (Castanotto and Rossi, 2009
). Given this promise and utility, many current investigations aim at better understanding the molecular mechanisms of RNAi and to find effective delivery methods for RNAi reagents.
There are several approaches to introduce silencing RNAs into cells. One of them is to directly introduce short interfering RNAs, 21-23 nucleotide duplexes targeting specific mRNAs, into the cells or tissue under investigation (Elbashir et al, 2001
). The disadvantage of this approach is that silencing is dependent on the amount of siRNA administered. A sustained silencing requires a constant and expensive siRNA supply. This shortcoming is eliminated when vectors containing sequences encoding short hairpin RNAs (shRNAs) are utilized (Paddison et al, 2002
). shRNAs are processed in the cells to produce siRNA. Cells transfected with these vectors can sustain RNAi-mediated gene silencing for 48 hours or longer under antibiotic selection.
The first large library of shRNA constructs targeting human and mouse genes was created in a retroviral vector, pShagMagic2 (pSM2) (Paddison et al, 2004
). This vector is subject to frequent recombination, does not contain a GFP marker and has inefficient viral packaging that limits the use in hard to transfect cells and in vivo
Aware of these problems, companies have transferred the shRNAs into more stable GFP-tagged lentiviral vectors that produce high-titer viruses (Moffat et al, 2006
). Lentiviruses are suitable for transduction of hard to transfect cell lines, primary cells and in vivo
applications. The latest generation of lentiviral constructs includes inducible shRNA production (TRIPZ Lentiviral Inducible shRNAmir Library®, www.openbiosystems.com
All the researchers who purchased the now outdated retroviral libraries cannot take advantage of these improvements. It is very expensive for laboratories to buy a complete lentiviral library, and the single lentiviral constructs range from $209 to $428. One inexpensive way to “upgrade” the shRNA without purchasing new ones is to sub-clone them to an appropriate lentiviral vector. Open Biosystems® provides a protocol for sub-cloning shRNA constructs from pSM2 into the lentiviral vector pGIPZ but it requires expensive kits and multiple steps. Therefore, if an investigator wants to sub-clone a high number of shRNA constructs, it may be cheaper and more convenient to just purchase the constructs.
We developed a protocol that greatly simplifies the transfer of shRNA sequences from the retroviral pSM2 vector into the pGIPZ lentiviral vector. The improvements in the protocol are reported in . This sub-cloning protocol can be applied to sub-clone shRNA from pSM2 to newer lentiviral vectors and possibly to sub-cloning schemes that involve plasmids and fragments of the same sizes reported here.
Table 1. Comparison between the Open Biosystems® and the proposed streamlined sub-cloning protocols for insert preparation. Cost- and time-savings are detailed for steps in insert preparation that are significantly different and are estimated for the smallest (more ...)