Sperm-mediated gene transfer studies on zebrafish in Singapore.

This paper describes the work conducted in the Department of Biological Sciences, National University of Singapore; on sperm-mediated gene transfer in zebrafish. This study began by carrying out direct interaction experiments of pUSVCAT reporter DNA with spermatozoa. Other constructs, including pXGH5, pMTL, pRSVL and pGEM-luc, were subsequently used. The different constructs were taken up by sperm cells with comparable efficiencies. In general, no reporter gene expression, Mendelian inheritance, or evidence of genomic integration of the foreign sequences were obtained. However, transmission of the reporter DNA through generations was observed. DNA uptake by sperm cells was shown using FISH and was enhanced by electroporation. The potential use of more recent approaches, such as REMI and ICSI are explored. Future research directions are discussed.

Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis.

The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.

Detection of diarrhetic shellfish poisoning toxins from tropical shellfish using liquid chromatography-selected reaction monitoring mass spectrometry.

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.

Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation.

Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000 or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one- to twofold by electroporation. Fertilization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non-electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non-mammalian sperm as in mammalian sperm.

Ultrastructural in situ hybridization and autoradiographic detection of foreign DNA in zebrafish spermatozoa.

Conditions have been established which permit specific hybridization of foreign DNA to complimentary DNA in ultra thin sections of zebrafish sperm prepared for electron microscopy (EM)1 after fixation in 2% paraformaldehyde, 2.5% glutaraldehyde and embedded in spurs resin. Zebrafish sperm incubated with foreign DNA were subjected to ultrastructural in situ hybridization and autoradiographic detection to determine if the sperm could retain and internalize the foreign DNA sequences. Specific and intense signals were detected from the sperm head indicating that the zebrafish sperm are capable of internalizing foreign DNA.

Assessment of pMTL construct for detection in vivo of luciferase expression and fate of the transgene in the zebrafish, Brachydanio rerio.

Early embryos of the zebrafish Brachydanio rerio were cytoplasmically microinjected with pMTL plasmid containing firefly luciferase gene, in both linearized- and supercoiled-plasmid forms, to evaluate in vivo expression, pattern of integration and germ-line transmission of the transgene in the host fish. It was possible to detect luciferase expression in vivo, and the pattern of time-course expression was similar in both linearized- and supercoiled-plasmid injected groups. Strong luciferase activity was detected 15-20 hours after injection, coinciding with early somitogenesis. Expression was detectable in a few 1 week-old individuals but was not detectable in all adults and in F1 progeny. In vivo screening for expression of the transgene in the developing embryo using luciferase assay as a method for detecting the presence of the transgenic fish compares favourably, with PCR and Southern blot analysis (SBA). No integration of the introduced DNA into the genome of treated fish and their progeny, was detected, instead it remained in extrachromosomal form. Most of the first generation founders were mosaic. Germline transmission was observed in one individual only. A probable reason for the absence of integration in this study when compared to the varying frequencies reported earlier in the same fish is discussed.

Gene transfer by microinjection in the zebra fish Brachydanio rerio.

Although transgenesis studies of fish started relatively later compared to those of mammals, they have not been completely neglected, and in recent years, many publications have appeared in this field. Reviews of transgenesis in fish are given by Maclean et al., Hew, Maclean and Penman, and Ozato et al. Much attention had been focused on commercially important food species, especially in the attempt to insert beneficial genes, such as the antifreeze and growth-hormone genes, into salmonids, such as trout and salmon. Zhang et al have successfully shown transfer, expression, and inheritance of rainbow trout growth-hormone cDNA microinjected into the common carp.

Ecology of marine fouling organisms at Eastern Johore Strait.

Marine biofouling causes problems to marine structure and obstructs condenser tubes in cooling systems which use sea water as the coolant. The main purpose of this study is to investigate the seasonal ecology of biofouling organisms such as the green mussel, Perna viridis, the dominant fouling species in the Eastern Johore Straits at the Senoko Power Station. The spawning time and its relationship with environmental conditions were studied. The physical, chemical and biological conditions of the sea at Senoko were monitored for a year. Settling slides were used to study the fouling succession in different monsoon seasons. The study showed that there were two main spawning peaks for the green mussel and that these peaks occurred during the intermonsoon months of November and April. These peaks were also correlated with the bimodal patterns for salinity, dissolved oxyen, bivalve veliger larval density and total plankton biomass of the Eastern Johore Strait water. Succession patterns were similar during the two monsoon seasons, however, the rate of fouling was probably greater during the southwest monsoon months. It is therefore advisable that the control or reduction of biofouling in Eastern Johore Strait should take into account the seasonal fluctuations and spawning of the fouling organisms.

Production of methyl farnesoate by the mandibular organs of the mud crab, Scylla serrata: validation of a radiochemical assay.

Parameters for the validation of a radiochemical assay to monitor the biosynthesis and release of methyl farnesoate (MF) by the mandibular organs (MO) of the mud crab, Scylla serrata, have been defined. On the basis of HPLC analysis, MF appeared to be the exclusive radiolabeled product of release, using [3H-methyl]methionine as precursor. HPLC of isooctane extracts of medium in which MO had been maintained similarly revealed that MF was the exclusive 3H-product and indicates that the isooctane partition assay can be employed to monitor MF release. The time course of MF biosynthesis and release suggested that the endogenous L-methionine pool is large, because both cumulative release and rate of release increased over an 8-hr incubation but remained constant between 8 and 16 hr. Glandular content of MF did not appear to stabilize until 6-8 hr after the start of the incubation. Cumulative MF release, as a function of glandular content or biosynthesis, increased gradually with time, but even after 16 hr the majority of biosynthesized MF was retained within the MO rather than released. L-Methionine concentration in the incubation medium influenced both MF biosynthesis and release, although the dose dependency was more apparent with biosynthesis. Although it was difficult to associate strictly a level of biosynthesis with L-methionine concentration, low rates of MF biosynthesis were observed at concentrations of less than 25 microM. Because high levels of biosynthesis were observed consistently between 60 and 70 microM L-met, all subsequent incubations employed a concentration of 65 microM. MO of S. serrata show a striking asymmetry in MF biosynthesis and release which is not time dependent. Hence the comparison of right and left glands in assessing the effects of experimental treatments is precluded.

Strontium/calcium ratio analysis of molluscan shells in Singapore waters using the X-ray fluorescence technique.

The Sr/Ca ratios in molluscan shells in Singapore waters were measured using the X-ray fluorescence (XRF) technique. Our results show that the Sr/Ca ratio varies greatly among different species. However, within the same species, this ratio is practically the same for samples collected from sites close together but varies significantly for samples from sites far apart. Furthermore, this study shows that whole shell analysis using the XRF technique is simple and quick and that its application to environmental monitoring seems feasible.