siRNA in Vivo

There are several ways to induce RNAi into a live host, but only three of them have consistently shown themselves effective in vivo: direct delivery of active siRNA constructs into animal tissue, delivery of short hairpin RNAs (shRNAs) through viral vectors, and delivery of RNAs through plasmid vectors. (These shRNAs are quickly processed into active siRNAs within the cell.) These methods have extended the applicability of siRNA technology to viral-based therapies and in vivo gene function experiments [1]. GenScript is proud to offer adenoviral, lentiviral, and retroviral vectors and a selection of proven siRNA plasmid vectors.

Creating knockout mice using transgenic siRNA:

The generation of knockout mouse lines, using conventional techniques, can be prohibitively expensive in both time and cost. Furthermore, many genes of interest cannot be knocked out completely without killing the mice. However, even difficult genes can be knocked down or out in live mice using transgenic siRNA [2]:

1. Design several vector-based siRNA constructs.
2. Test the vector-based siRNA in cell lines such as HEK293 to confirm that they downregulate the expression of the gene of interest.
3. Linearize and electroporate the vector-based siRNA into ES cells. Transfected ES cell lines can be selected via drug resistance.
4. Generate the transgenic mouse line using the drug-resistant ES cell lines.

 

Advantages of transgenic siRNA over conventional knockout technology:

• Transgenic siRNA is a time-saving and cost-effective approach to the generation of knockout mice. The time and expense needed to build siRNA constructs (three weeks and $375 per construct) is only a fraction of that of conventional methods (three months and at least $8000).
• Transgenic siRNA does not require genomic sequencing, which is essential for conventional knockout technology.
• Transgenic siRNA can generate both partial and complete knockout phenotypes, allowing for the knockout of difficult or vitally necessary genes.

 

Introducing siRNA into animals via other methods:

• Plasmid injection: Vector-based siRNA in plasmid form can be directly injected into certain organs [3].
• Tail vein: Vector-based siRNA can be introduced into various animals using tail vein hydrodynamic injection [4].
• Liposome formulation: Vector-based siRNA can be formulated into cationic liposomes and introduced into animals via intravenous injection [5].

 

References:

1. Haibin Xia, Qinwen Mao, Henry L. Paulson, Beverly L. Davidson. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol. 2002 Oct; 20 (10): 1006-1010.
2. Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol. 2003 May; 21 (5): 559-561.
3. Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther. 2003 Nov; 8 (5): 769-776.
4. Gratsch TE, De Boer LS, O'Shea KS. RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos. Genesis. 2003 Sep; 37 (1): 12-17.
5. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 2003 Apr 4; 327 (4): 761-766.