Methods of In Vivo Gene-Specific Knockdown Using Morpholino and Vivo-Morpholino Oligonucleotides

Resumo

The functioning of the small GTPase gene, Ras-dva1, has been studied during regeneration processes of the tadpole tails of the clawed frog Xenopus laevis. For this purpose, we have developed two approaches for the gene knockdown using injections of sequence-specific antisense morpholino oligonucleotides (MO) or vivo-morpholino oligonucleotides (vivo-MO). It has been shown for the first time that intracellular Ras-dva-specific MO injected into Xenopus 4–16 of blastomere embryos or vivo-MO injected directly into the distal part of the tadpole tail at stages 40–41 efficiently inhibit the Ras-dva1 gene functioning and leads to the same morphological malformations of the tail regeneration. Using qRT-PCR, we detected the inhibition of expression of the regeneration marker genes (Fgf20a and Msx1b) on the first day after amputation in the tail tissues of tadpoles with the Ras-dva1 knockdown.