The impact and potential of Marine biotechnology, across a wide spectrum of applications ranging from the environment to biomedicines is well-known. Economically, the field of marine biotechnology is still on the improving side. Researchers have proposed discovery programs for certain key areas of marine biotech in order to increase the yield and application of marine bio-products. The areas where improvements could substantially impact drug discovery and development are:
(1) access to new marine product sources;
(2) meet the supply and demand of drug discovery and development process;
(3) enhancing paradigms for screening and discovering potential marine bio-products;
(4) understanding biological mechanisms of marine bio-products; and
(5) streamlining marine bio-product development process regulations.
The ocean is immeasurably rich in chemical and biological diversity. It encompasses more than 70 percent of earth's land and is inhabited by more than 40,000 described species of animals and plants. It was reported that a relatively less number of marine animals, microbes, and plants have successfully yielded more than 10,000 novel chemicals.
Some of the commercially available marine bio-products and their applications include:
(1) Aequorin, extracted from Aequora victoria (bioluminescent jellyfish), acts as a bioluminescent calcium indicator;
(2) Ara A (acyclovir), extracted from Cryptotethya cyrta (marine sponge), is used as an antiviral drug for herpes infections; and
(3) Ara-C (cytosar-U), used as an anticancer drug for treating leukemia.
Molecular approaches have offered potential alternatives to supply natural products (via gene identification, cloning, isolation, and heterologous expression) and to discover molecular diversity sources (via gene identification and biosynthetic pathways).
Manipulating heterologously expressed biosynthetic genes (secondary metabolite) produces novel compounds that have a potential pharmaceutical value.
The screening process to identify natural substances for biologically active compounds has seen radical changes in recent years. With the introduction of high-throughput-screening (HTS) technology, more than 50,000 materials can be screened for a specific biochemical or biological property in less time; say, around 3 to 4 months. Natural marine product extracts or mixtures must be purified and immediately screened for their components.
Transgenic Fish and Methods Involved
Transgenic fish are produced by injecting plasmid DNA into the fish eggs. Since the pronucleus of a fertilized egg is not visible in a zebra fish, DNA is injected into either the yolk or the cytoplasm. The cytoplasmically injected DNA integrates into the genome and is transmitted through the germ-line. The embryonic cell division occurs rapidly since DNA is injected into the cytoplasm of the egg. Different methods were introduced to improve the efficacy of the transgene integration into the genome of the host.
The methods include:
(1) using pseudo-typed virus;
(2) integration of transgene mediated by retroviral integrase protein;
(3) integration through transposable elements; and
(4) DNA binding to nuclear proteins. Nuclear signal localization (NLS) aims to facilitate the transgene DNA uptake by the host nucleus. NLS binding to the DNA enhances the zebrafish embryo nuclei uptake thereby increasing the frequencies of transgene integration and expression and germline transmission.
There are several systems to investigate the nucleocytoplasmic transport. Some of them are:
(1) NLSs binds to the DNA: the NLSs specificity of nuclear import along with the peptide positive charge makes the NLS a good candidate for plasmid DNA binding. Synthetic NLS analogous (of SV40 T antigen) were bound to a plasmid that carried a luciferase reported gene;
(2) NLS-DNA complexes imported to embryo by the nuclei: This procedure is based on blastomere fractionation. It was described to separate the embryonic nuclei of a zebrafish from cytoplasmic fractions. With an isolated nucleus, it is possible conduct a PCR examination on the presence of the DNA injected in the nuclear fraction using specific primers that amplify the fragment of the injected DNA;
(3) NLS-DNA complexes targeted in vitro: An in vitro assay was developed to examine the conditions that promote the transgene DNA nuclear import. This procedure consists of from sea urchin pronuclei that were incubated in a fertilized egg extract (zebrafish) that contained NLS-DNA complexes. The extract showed two things: the NLS- DNA bind to nuclear membrane in ATP's absence; and translocation of complexes with ATP addition.
The biggest advantage of NLS peptides is the quick transfer of transgene DNA to the nucleus from the cytoplasm. This is particularly important when working with transgene integration during embryo development process.
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