Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish thrombocytes

Abstract

Morpholino and vivo-morpholino gene knockdown methods have been used to study thrombocyte function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study thrombocyte function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to thrombocyte functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study thrombocyte function in zebrafish.

Introduction

Antisense deoxyoligonucleotides have previously been used to successfully inhibit gene function [1], [2]. Unfortunately, nuclease degradation of these single-stranded standard oligonucleotides (SOs) results in a short half-life for these SOs and has prompted subsequent designs of modified oligonucleotides, such as phosphorothioates [3] and morpholino oligonucleotides (MOs) [4]. However, cell culture models have indicated that phosphorothioates have a limited ability to enter cells and upon entry have caused cell toxicity [5], [6], [7]. In contrast, studies of MOs injected into 1–8 cell stage zebrafish embryos have demonstrated that MOs easily diffuse into cells via cytoplasmic bridges, were not toxic, and suppressed protein expression either by altering the splicing mechanism or by translational blocking [8], [9]. Thus, these MOs have been commonly used to study gene function in the zebrafish model. However, since this technology is only effective for approximately 8 days post injection, and since it is difficult to study biochemical pathways of thrombocyte function in embryos after knockdown, it was essential to find a more effective method to study their function in adult zebrafish. At that time, the newly available vivo-morpholino (VMO) had been shown to enter into tissues, resulting in successful knockdowns of gene function in adult mice [10], [11]. In this VMO method, dendrimeric octaguanidine moieties are conjugated to MOs. We introduced the application of this VMO technology to adult zebrafish and were successful in the knockdown of αIIb [12]. Since then, a number of gene knockdowns have been performed in adult zebrafish with efficiencies no less than 50% [13], [14], [15], [16], [17], [18], [19]. However, custom-ordering multiple VMOs to study thrombocyte function via genome-wide knockdowns would be considerably expensive. Thus, we hypothesized that if we could create a hybrid that could easily enter cells by piggybacking an SO antisense to a target mRNA sequence onto a readily available control VMO (cVMO), then this hybrid could bind to the target mRNA leading to mRNA degradation and resulting in gene knockdown and subsequent suppression of protein expression. In this paper, proof-of-principle was demonstrated by knockdown of the αIIb gene since this gene has been established as a target to validate knockdown technology in studies involving thrombocyte function [12], [20].