Establishing a Working Protocol for Plasmid Cloning and shRNA Design in Endogenous Brachionus manjavacas Gene TRP7

Abstract

Current transfection protocol in rotifers only allows for temporary transfection within rotifers and does not allow for the continuous knockdown of endogenous genes, thereby inhibiting the possibility of observing long-term biological effects in response to specific perpetual gene knockdowns. This study aims to address this particular issue by establishing a working protocol for plasmid cloning and shRNA design within an endogenous gene of B. manjavacas with known biological effects, allowing for the exploration into the optimization of a transfection protocol and demonstration of RNAi knockdown of the known gene within the rotifers as subsequent studies. Manipulation of gene expression in rotifers could occur through plasmid vector insertions, which induce silencing of a gene’s expression with short hairpin RNA (shRNA), via RNAi. This would effectively stimulate gene knockdown, allowing for the observation of biological effects such as changes in fecundity and lifespan. With the establishment of a working protocol for plasmid cloning and shRNA design, as a result of this study, the optimization of a transfection protocol for rotifers is explored. With increased efficiency in the transfection of rotifers, populations of rotifers expressing the plasmid can be amassed, allowing for experimental design that examine the varying aging mechanisms and effects that are stimulated due to permanent changes in target gene expression through RNAi. This, in turn, could give rise to the identification of evolutionarily conserved genes that regulate organismal aging, which could lead to further implications in the field of pharmacological intervention in mammalian aging as well as in the field of biogerontology overall.