18] Use of antisense oligonucleotides in human neuronal and astrocytic cultures

Abstract

Antisense oligodeoxynucleotides (ODNs) can potentially block the expression of a single gene in living cells by binding to a complementary messenger ribonucleic acid (mRNA). These molecules can enter cultured cells, recognize a specific gene sequence, and inhibit its expression. For this reason, antisense oligodeoxynucleotide strategies are largely used to understand specific gene functions in biological processes and during development. Several factors must be considered when treating cells with antisense and appropriate controls, which must be chosen to demonstrate that the effects of treatment are due to specific gene inhibition; there are many nonspecific effects that are not due to antisense mechanisms alone. This chapter describes the standard protocols for antisense treatment of human fetal brain cultures and for controls that are required to interpret results. An antisense molecule can be considered specific if it determines a significant decrease in target RNA and related protein levels and if there is no strong loss of cell viability. The interaction between antisense and RNA requires a minimum level of affinity.