6-3.3Methods for creating transgenic animals:

With the advances of recombinant DNA technology, different methods have been developed for generating transgenic animals. The  examples of commonly used methods applied in transgenic animal production are-

• Gene knockdown using  RNA interference (RNAi),
• Retroviral mediated gene transfer
• Embryonic stem cell technology
• DNA microinjection
• Nuclear transfer
• Sperm as vector

6-3.3.1Gene knockdown using RNA interference (RNAi):

RNA interference (RNAi) induced in mammalian cells by introducing antisense RNA to generate double stranded RNA (dsRNA) structure (or hairpin) which are cleaved to generate small fragments of RNA (~22bp length). Double stranded RNA are indentified and cleaved by Dicer enzyme to generate small interfering RNA (siRNA) or micro RNA (miRNA). miRNA is single stranded RNA molecule which inhibits translation of mRNA whereas siRNA are double stranded RNA which cleave RNA. A riboprotein complex is generated by the small RNA fragments called RISC (RNA induced silencing complex) which binds to homologous mRNA molecule and initiate the cleavage, thus silencing the gene expression. (Fig6-3.3.1)

In mammalian cells, RNAi effect can be introduced by generating small interfering RNA (siRNA) or small hairpin RNA (shRNA). Using this approach, several transgenic mice and rat models have been developed for potential medical and pharmaceutical applications.

RNAi technology has several advantages over other approaches. Once stably established RNAi can be synthesized directly inside the mammalian cells thus avoiding any other cloning steps.  RNAi approach can be designed to regulate gene expression either transcription or translation stage.