A ribozyme-mediated, gene "knockdown" strategy for the identification of gene function in zebrafish

Abstract

The zebrafish system offers many unique opportunities for the study of molecular biology. To date, only random mutagenesis, and not directed gene knockouts, have been demonstrated in this system. To more fully develop the potential of the zebrafish system, an approach to effectively inhibit the expression of any targeted gene in the developing zebrafish embryo has been developed. This approach uses a transient, cytoplasmic, T7 expression system, injected into the fertilized zebrafish egg to rapidly produce high levels of a ribozyme directed against the mRNA encoded by the targeted gene to inhibit its expression. In a demonstration of this strategy, expression of the recessive dominant zebrafish no tail gene was effectively inhibited by using this strategy to yield a phenotype identical to that resulting from a known defective mutation in this same gene. This, ribozyme-mediated, message deletion strategy may have use in determining the function of genetic coding sequences of unknown function.

Figure 1

The ntl ribozyme expression vector and schematic secondary structure of ntl ribozyme RNA. (A) Ntl ribozyme expression vector pT₇vaRz. The vector pT₇vaRz(ntl) was constructed first by an insertion of a ribozyme sequence (Rz) against the ntl mRNA into a va I RNA expression cassette; the cassette containing the ribozyme sequence (va-Rz-loop-va’) subsequently was cloned into a pTM-1 expression vector (27) lacking the encephalomyocarditis virus internal ribosome entry site sequence in such a way that the expression of Rz is under the control of both a T₇ promoter (P_T7) and an adenovirus va I internal promoter, a pol III promoter (upstream va). The P_T7-driven transcription stops at the T₇ terminator (T_T7) whereas the va I-driven transcription terminates at the 3′ end of the loop. (B) The sequences flanking Rz(ntl) were designed in such a way that the 5′ and 3′ ends of the ntl ribozyme form a stable stem structure (18). Both the ribozyme and its targeted, complementary ntl mRNA sequences, as well as the cleavage site (435), also are shown.

Figure 2

Phenotypic changes after development of zebrafish eggs that had been injected with pT₇vaRz(ntl)₄₃₅, pT₇T₇, and T7 RNA polymerase. (A) Normal zebrafish at 96 h of embryogenesis. (B) Two examples of partial no tail phenotypic changes at 96 h of embryogenesis in zebrafish developing from eggs injected with pT₇vaRz(ntl)_435, pT₇T₇, and T7 RNA polymerase. (C) An example of full no tail phenotypic changes at 96 h of embryogenesis in zebrafish developing from eggs injected with pT₇vaRz(ntl)_435, pT₇T₇, and T7 RNA polymerase. (D) A homozygous no tail mutant zebrafish (ntl^b195) (23) at 96 h of embryogenesis.

Figure 3

Northern blot analyses of the reduction of ntl mRNA and the presence of ribozyme in the pT7vaRz(ntl)₄₃₅-injected zebrafish embryos. Fertilized zebrafish eggs were injected with pT7vaRz(ntl)_435, pT₇T₇, and T7 RNA polymerase as described in Fig. 2. Total RNA was isolated from 50 injected and uninjected embryos at 5.2, 12, and 24 h postfertilization. Each RNA sample (8 μg) was subjected to electrophoresis in a 1% agarose–formaldehyde gel. After gel transfer, the membrane containing the transferred RNAs was hybridized with appropriate probes as described in Materials and Methods. (A) Northern blot with the ntl probe. Rz, RNA isolated from the injected embryos; C, RNA isolated from uninjected embryos. (B) The same membrane rehybridized with ribozyme and actin probes. The actin mRNAs serve as the internal RNA quantitation references for each time point.