Biomedical subjects

Surintorn Boonanuntanasarn

Publications and source records attributed to Surintorn Boonanuntanasarn.

4 recordsLinked to original sources

PubMed2005

High-efficiency gene knockdown using chimeric ribozymes in fish embryos.

We report an effective gene knockdown technique in rainbow trout embryos using additional RNA components combined with ribozymes (R(z)s). Chimeric R(z)s (tR(z)Cs) containing tRNA(Val), R(z) against GFP, and a constitutive transport element were microinjected into transgenic embryos. tR(z)Cs induced greater gene interference than R(z)s alone. Control tR(z)Cs did not affect unpaired bases of target RNA, and the tR(z)C did not interfere with non-relevant gene expression, suggesting that the tR(z)C-mediated gene-interference effects were sequence-specific. Furthermore, the tR(z)C-containing expression vector specifically suppressed target GFP expression in transgenic trout. tR(z)Cs enhance R(z) cleavage and could therefore be powerful tools for studying unknown gene function in vertebrates.

Animals↗

PubMed2004

Molecular cloning, gene expression in albino mutants and gene knockdown studies of tyrosinase mRNA in rainbow trout.

Tyrosinase has a role in melanin synthesis and several defects of the tyrosinase gene lead to albinism. Here, we cloned and characterized rainbow trout tyrosinase cDNAs and carried out the molecular and biochemical characterization of albino mutants. Two types of cDNA were cloned: tyrosinase-1 (Tyr-1) and tyrosinase-2 (Tyr-2). Both contained regions predicted to encode structural features of tyrosinase, and phylogenetic analysis confirmed that Tyr-1 and Tyr-2 were members of the tyrosinase family. Tyr-1 transcripts were first detected in embryos at 5 d post-fertilization (dpf) and Tyr-2 transcripts at 15 dpf. 3,4-dihydroxyphenylalanine assays revealed significantly reduced tyrosinase activities in dominant and recessive albino mutants compared with wild-type embryos. However, reverse-transcription PCR showed no differences in the amounts or lengths of the coding regions of Tyr-1 and Tyr-2 transcripts between wild-type embryos and albino mutants. Antisense morpholino oligonucleotides (AMOs) designed to knockdown tyrosinase gene expression in wild-type embryos led to reduced pigmentation in the retina and skin of embryos at 25 and 35 dpf, respectively. Furthermore, the tyrosinase activities of AMO-treated embryos were significantly reduced. We conclude that both Tyr-1 and Tyr-2 are crucial for melanin synthesis in rainbow trout embryos. Furthermore, we describe a potential application of AMOs in the treatment of hyperpigmentation.

Albinism↗

PubMed2003

Specific gene silencing using small interfering RNAs in fish embryos.

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.

Animals↗

PubMed2002

Gene knock-down in rainbow trout embryos using antisense morpholino phosphorodiamidate oligonucleotides.

Gene knock-down technology using antisense molecules has many applications for studying gene function, disrupting undesirable genetic traits, as well as providing effective therapy for a number of viral diseases. Encouraged by these applications, we developed a gene knock-down technique to interfere with gene expression using transgenic rainbow trout expressing the green fluorescent protein (GFP) gene as a model. One of the antisense morpholino phosphorodiamidate oligonucleotides (AMOs) used in this study (AtGFP-1) was 25 nucleotides in length and localized against codons 2 to 8 of GFP messenger RNA. Microinjection of AtGFP-1 into the blastodisc of fertilized eggs decreased the level of GFP gene expression in a dose-dependent manner, A comparison of the effects of various doses of AtGFP-1 suggested that 10 ng of AtGFP-1 was the optimal concentration in that it interfered with specific gene expression without being strongly toxic to trout embryos. Conversely, morpholino phosphorodiamidate oligonucleotides with the inverted AtGFP-1 sequence, which cannot bind to the target mRNA, did not inhibit GFP gene expression. AtGFP-1 did not affect the expression of nontargeted genes such as the skeletal muscle actin and foreign lacZ genes. These results also indicate that AtGFP-1 interfered with the expression of only the targeted gene. Western blot and reverse transcriptase polymerase chain reaction analyses revealed that the amount of GFP protein drastically decreased whereas the mRNA level was not affected by AtGFP-1, suggesting that AtGFP-1 blocked specific gene function at the translational level. Further, this gene inhibition persisted until the hatching stage. Another AMO, which was localized against the junction region between the 5? untranslated region and the starting codon of GFP mRNA (AtGFP-2), also caused inhibition effects. Thus AMOs can have potent and specific gene knock-down effects in trout embryos. This technology may be useful for examining the roles of selected genes and disrupting their expression during embryonic development of salmonid fish.

Journal Article↗