Rapid method for identification of transgenic fish zygosity

Identification of zygosity in transgenik fish is normally achieved by PCR analysis with genomic DNA template extracted from the tissue of progenies which are derived by mating the transgenic fish and wild-type counterpart.  This method needs relatively large amounts of fish material and is time- and labor-intensive. New approaches addressing this problem could be of great help for fish biotechnologists.  In this experiment, we applied a quantitative real-time PCR (qr-PCR) method to analyze zygosity in a stable line of transgenic zebrafish (Danio rerio) carrying masu salmon, Oncorhynchus masou D6-desaturase-like gene. The qr-PCR was performed using iQ SYBR Green Supermix in the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, USA).  Data were analyzed using the comparative cycle threshold method.  The results demonstrated a clear-cut identification of all transgenic fish (n=20) classified as a homozygous or heterozygous.  Mating of those fish with wild-type had revealed transgene transmission to the offspring following expected Mendelian laws. Thus, we found that the qTR-PCR to be effective for a rapid and precise determination of zygosity in transgenic fish. This technique could be useful in the establishment of breeding programs for mass transgenic fish production and in experiments in which zygosity effect could have a functional impact. Keywords: quantitative real-time PCR; zygosity; transgenic fish; mass production   ABSTRAK Identifikasi sigositas ikan transgenik biasanya dilakukan menggunakan analisa PCR dengan cetakan DNA genomik yang diekstraksi dari jaringan ikan hasil persilangan antara ikan transgenik dan ikan normal.   Metode ini memerlukan ikan dalam jumlah yang banyak, dan juga waktu serta tenaga.  Pendekatan baru untuk mengatasi masalah tersebut akan memberikan manfaat besar kepada peneliti bioteknologi perikanan.  Pada penelitian ini, kami menggunakan metode PCR real-time kuantitatif (krt-PCR) untuk menganalisa sigositas pada satu strain ikan zebra (Danio rerio) transgenik yang membawa gen D6-desaturase-like dari ikan salmon masu, Oncorhynchus masou.  krt-PCR dilakukan menggunakan iQ SYBR Green Supermix pada mesin iCycler iQ Real-time PCR Detection system (Bio-Rad Laboratories, USA).  Data dianalisis menggunakan metode pembandingan nilai cycle threshold.  Hasil penelitian menunjukkan bahwa semua ikan transgenik (n=20) yang diidentifikasi dapat diklasifikasikan secara jelas sebagai ikan homosigot atau heterosigot.  Persilangan antara ikan transgenik tersebut dengan ikan normal menunjukkan transmisi transgen ke keturunannya mengikuti hukum segregasi Mendel.  Dengan demikian, metode krt-PCR adalah efektif untuk penentuan sigositas secara cepat dan tepat pada ikan transgenik.  Teknik ini dapat berguna dalam program produksi ikan transgenik secara massal dan dalam percobaan dimana faktor sigositas memberikan pengaruh nyata. Kata kunci: kuantitatif real-time PCR; sigositas, ikan transgenik; produksi massa

Effectiveness of hCMV, mEF1a and mAct promoters on driving of foreign gene expression in transgenic zebrafish

Highly unsaturated fatty acids (HUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have long been recognized for its beneficial effect for human health and development.   The D6 fatty acid desaturase is generally considered to be the rate-limiting factor in HUFA biosynthesis.  Here, as the first step of study, we conducted experiment to select an appropriate construct that allows higher expression levels of masu salmon (Oncorhynchus masou) D6-desaturase gene in zebrafish (Danio rerio) in order to increase its activity for synthesizing EPA/DHA.  Salmon D6-desaturase cDNA (sD6) was separately ligated with human cytomegalovirus (hCMV), medaka elongation factor 1a (mEF1a) and medaka b-actin (mAct) promoters.  The resulted construct was designated as hCMV-sD6, mEF1a-sD6 and mAct-sD6, respectively.  Each of the constructs in circular DNA form was microinjected into 1-cell stage embryos at a concentration of 30mg/ml. Transgenic individuals were identified by polymerase chain reaction (PCR) and their expression levels were analyzed by reverse transcription PCR.  The first (F1) and second (F2) generation was produced by crossing the transgenic founder F0 and F1, respectively, with wild-type fish.  The results showed that the highest transient gene expression level was obtained from the mAct-D6 construct, followed respectively by EF1a-D6 and hCMV-D6 construct. The transmission rate of transgene into F1 generation was 4.2%-44.1%, and into F2 was followed the Mendellian segregation pattern.   Expression of transgene in F2 generation was varied between strains regarding as the mosaics of F0 fish.  Now, a transgenic system to study the modification of fatty acid biosynthesis pathways in fish was established.  Further investigations are to produce fish containing higher levels of EPA and DHA. Keywords: desaturase, nutraceutical fatty acid, transgenic, zebrafish, masu salmon   Abstrak Promoter merupakan regulator yang menentukan tempat, waktu dan tingkat ekspresi gen.  Pada penelitian ini, kami melakukan seleksi kontruksi plasmid yang tepat yang menghasilkan tingkat ekspresi yang tinggi dari gen D6-desaturase-like ikan masu salmon (Oncorhynchus masou) yang ditransfer ke ikan zebra (Danio rerio) untuk meningkatkan kemampuannya dalam mensintesa EPA/DHA.  cDNA D6-desaturase-like (OmD6FAD) dari ikan salmon masu diligasi secara terpisah dengan promoter dari cytomegalovirus manusia (hCMV), elongation factor 1a (mEF1a) dan b-actin (mbAct) dari ikan medaka, untuk membuat konstruksi plasmid yang berturut-turut disebut sebagai hCMV-OmD6FAD, mEF1a- OmD6FAD dan mbAct-OmD6FAD. Konstruksi tersebut dengan konsentrasi 30mg/ml disuntikkan ke embrio pada saat fase satu sel. Individu transgenik diidentifikasi menggunakan PCR dan tingkat ekspresi transgen dianalisa dengan RT-PCR.   Hasil menunjukkan bahwa tingkat ekspresi sementara yang tertinggi dari gen asing adalah diperoleh dari konstruksi mbAct-OmD6FAD, diikuti selanjutnya oleh EF1a-OmD6FAD dan hCMV- OmD6FAD. Transgen telah ditransmisikan ke ikan generasi F2 dengan mengikuti pola segregasi Mendel. Tingkat ekspresi transgen yang tinggi pada jaringan ikan F2 yang diperiksa telah diperoleh.  Dengan demikian, sebuah sistem transgenik untuk memodifikasi biosistesa asam lemak pada ikan telah dikembangkan.  Kata kunci: promoter, desaturase asam lemak, transgenik, ikan zebra, ikan salmon mas

Aplication of Gene Transfer in Aquaculture

Recently, global food security has become a hot issue by the public in national as well as out of the country. Aquacultural output will need to be increased several fold in order to meet the rising demands for fish in coming years as the increasing of mankind population. The intensity and capacity of production is expected to increase using biotechnology approach. One of the advances biotechnologies that expected to be a powerful approach for aquaculture development is transgenic technique. This technique has been applied to several commercially valuable species. This review describes various techniques of gene transfer, persistence and expression of transferred gene, application and future aspect of gene transfer research in aquaculture. Key words: Gene transfer, gene expression, biotechnology,  aquaculture   ABSTRAK Saat ini, keamanan pangan telah menjadi isu hangat di masyarakat baik di dalam maupun di luar negeri. Produksi akuakultur diharapkan dapat ditingkatkan beberapa kali lipat untuk memenuhi kebutuhan pangan berupa ikan dimasa-masa mendatang akibat peningkatan populasi manusia. Intensitas dan kapasitas produksi diharapkan meningkat dengan menggunakan pendekatan bioteknologi. Salah satu teknik modern yang diduga akan menjadi sarana yang berguna dalam pengembangan akuakultur adalah teknologi transfer gen. Teknik ini telah diaplikasikan pada spesies-spesies yang memiliki nilai ekonomis. Ulasan ini menggambarkan variasi metode transfer gen, persistensi dan ekspressi dari gen yang ditransfer, aplikasi dan prospeknya ke depan dari penelitian transfer gen dalam akuakultur. Kata kunci : transfer gen, ekspressi gen, bioteknologi, akuakultu

Nature of the Electronic Excitations near the Brillouin Zone Boundary of Bi2_2Sr2_2CaCu2_2O8+δ_{8+\delta}

Based on angle resolved photoemission spectra measured on different systems at different dopings, momenta and photon energies, we show that the anomalously large spectral linewidth in the $(\pi,0)$ region of optimal doped and underdoped Bi$_2$Sr$_2$CaCu$_2$O$_{8+\delta}$ has significant contributions from the bilayer splitting, and that the scattering rate in this region is considerably smaller than previously estimated. This new picture of the electronic excitation near $(\pi,0)$ puts additional experimental constraints on various microscopic theories and data analysis.Comment: 5 pages, 4 figure

Effect of anisotropic impurity scattering in superconductors

We discuss the weak-coupling BCS theory of a superconductor with the impurities, accounting for their anisotropic momentum-dependent potential. The impurity scattering process is considered in the t-matrix approximation and its influence on the superconducting critical temperature is studied in the Born and unitary limit for a d- and (d+s)-wave superconductors. We observe a significant dependence of the pair-breaking strength on the symmetry of the scattering potential and classify the impurity potentials according to their ability to alter T_c. A good agreement with the experimental data for Zn doping and oxygen irradiation in the overdoped cuprates is found.Comment: 31 pages, RevTex, 15 PostScript figure

Evidence for an energy scale for quasiparticle dispersion in Bi_2Sr_2CaCu_2O_8

Quasiparticle dispersion in $Bi_{2}Sr_{2}CaCu_{2}O_{8}$ is investigated with improved angular resolution as a function of temperature and doping. Unlike the linear dispersion predicted by the band calculation, the data show a sharp break in dispersion at $50\pm10$ $meV$ binding energy where the velocity changes by a factor of two or more. This change provides an energy scale in the quasiparticle self-energy. This break in dispersion is evident at and away from the d-wave node line, but the magnitude of the dispersion change decreases with temperature and with increasing doping.Comment: 4 figure

Prevalence and impact of combined vision and hearing (dual sensory) impairment : a scoping review

Funding: MJB is supported by the Wellcome Trust (207472/Z/17/Z). JR's appointment at the University of Auckland is funded by the Buchanan Charitable Foundation, New Zealand. TB is funded by Christian Blind Mission (CBM International). The Lancet Global Health Commission on Global Eye Health is supported by The Queen Elizabeth Diamond Jubilee Trust, Moorfields Eye Charity [grant number GR001061], NIHR Moorfields Biomedical Research Centre, Wellcome Trust, Sightsavers, The Fred Hollows Foundation, The SEVA Foundation, British Council for the Prevention of Blindness and Christian Blind Mission. CGBJ is supported by an NHMRC Fellowship (GNT 1142897) and a WA Future Health Research and Innovation Fund Fellowship.Hearing and vision impairments are common globally. They are often considered separately in research, and in planning and delivering services. However, they can occur concurrently, termed dual sensory impairment (DSI). The prevalence and impact of hearing and vision impairment have been well-examined, but there has been much less consideration of DSI. The aim of this scoping review was to determine the nature and extent of the evidence on prevalence and impact of DSI. Three databases were searched: MEDLINE, Embase and Global Health (April 2022). We included primary studies and systematic reviews reporting the prevalence or impact of DSI. No limits were placed on age, publication dates, or country. Only studies where the full text was available in English were included. Two reviewers independently screened titles, abstract, full texts. Data were charted by two reviewers independently using a pre-piloted form. The review identified 183 reports of 153 unique primary studies and 14 review articles. Most evidence came from high-income countries (86% of reports). Prevalence varied across reports, as did age groups of participants and definitions used. The prevalence of DSI increased with age. Impact was examined across three broad groups of outcomes-psychosocial, participation, and physical health. There was a strong trend towards poorer outcomes for people with DSI across all categories compared to people with one or neither impairment, including activities of daily living (worse for people with DSI in 78% of reports) and depression (68%). This scoping review highlights that DSI is a relatively common condition with substantial impact, particularly among older adults. There is a gap in evidence from low and middle-income countries. There is a pressing need for a consensus position on the definition(s) of DSI and standardisation of reporting age groups to enable reliable estimates to be ascertained and compared and responsive services developed.Publisher PDFPeer reviewe

Signature of Superfluid Density in the Single-Particle Excitation Spectrum of Bi2Sr2CaCu2O8+delta

We report that the doping and temperature dependence of photoemission spectra near the Brillouin zone boundary of Bi2Sr2CaCu2O8+delta exhibit unexpected sensitivity to the superfluid density. In the superconducting state, the photoemission peak intensity as a function of doping scales with the superfluid density and the condensation energy. As a function of temperature, the peak intensity shows an abrupt behavior near the superconducting phase transition temperature where phase coherence sets in, rather than near the temperature where the gap opens. This anomalous manifestation of collective effects in single-particle spectroscopy raises important questions concerning the mechanism of high-temperature superconductivity.Comment: 14 pages, open with Acrobat 3.0+. "Science" 280, (July, 2000) 277, submitted 11 April 200