Evaluation of Nutritive Value of Raw and Fermented De-oiled Physic Nut, Jatropha curcas Seed Meal in the Formulated Diets for Rohu, Labeo rohita (Hamilton) Fingerlings

Rohu (Labeo rohita) fingerlings, were fed de-oiled Jatropha curcas seed meal (DJSM) supplemented diets for 60 days and the effectiveness, if any, on the growth was evaluated. Nine isonitrogenous (35 % crude protein) and isocaloric (4.0 kcal g^?1) diets were formulated of which one was reference diet (RD, fishmeal based control diet) and the other eight were experimental diets prepared by incorporating raw (D1–D4) and fermented (D5–D8) DJSM at 10, 20, 30 and 40 % levels by weight, respectively. Autoclaved DJSM was processed through solid state fermentation (SSF) for 15 days at 37 ± 1 °C by an exo-enzyme producing bacterium, Bacillus cereus Lr.H.23 isolated from the hindgut of rohu, L. rohita. Processing through SSF caused decrease in the contents of crude fibre and anti-nutritional factors, but increase in the levels of free amino acids and free fatty acids. In terms of growth performance, feed utilization efficiency and apparent protein digestibility, fish fed diet D7 containing 30 % fermented DJSM showed the best performance, which differed significantly (P < 0.05) from that of the fish fed diets containing raw DJSM. The results indicated that an inclusion level up to 30 % fermented DJSM replacing 15 % FM in the practical diet for rohu fingerlings can be proposed when compared to the RD. However, further experiments are required to recommend the ingredient for use in industry.     

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70–80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r ²?=?0.946–0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.     

The promoter of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, Bombyx mori

The fat body plays multiple, crucial roles in the life of silkworms. Targeted expression of transgenes in the fat body of the silkworm, Bombyx mori, is important not only for clarifying the function of endogenous genes expressed in this tissue, but also for producing valuable recombinant proteins. However, fat body-specific gene expression remains difficult due to a lack of suitable tissue-specific promoters. Here we report the isolation of the fat body-specific promoter of Bmlp3, a member of the 30K protein family of silkworms. The 1.1 kb fragment from ?374 to +738 of Bmlp3 displayed strong promoter activity in the cell lines BmE and Spli-221. In transgenic silkworms, a DsRed reporter gene controlled by the 1.1 kb Bmlp3 promoter fragment was expressed specifically in the fat body in a stage-specific pattern that was nearly identical to the endogenous Bmlp3 gene. We conclude that the 1.1 kb Bmlp3 promoter fragment is sufficient to direct tissue- and stage-specific expression of transgenes in the fat body of silkworms, highlighting the potential use of this promoter for both functional genomics research and biotechnology applications.     

Development and characterization of a continuous macrophage cell line,LRTM, derived from thymus of Labeo rohita (Hamilton 1822)

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco’s modified Eagle’s medium at 37°C with 5% CO₂ and showed 85+?% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.     

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78

Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3 × 10⁷ cfu bacteria per 20 g of body weight intraperitoneally. The expression of these genes was studied in infected liver, anterior kidney, and spleen tissues of rohu at different time periods: 0, 1, 3, 6, 12, 24, 48, 72 h, 7, and 15 days post-infection by qPCR. The Hsp gene modulation was greater in liver as compared to spleen and kidney tissues. Induced expression of Apg2, Hsp90, Grp78, Grp75, and Hsc70 was noticed during peak periods (3 to 24 h post-challenge) of bacterial infectivity. Hsp70 was found to be down-regulated during the process of infection. In contrast to the other six genes, Hsp30 showed a variable expression pattern in all three tissues. Grp78 was found to be the most highly (1,587-fold) expressed gene in liver at 12 h post-challenge. Further, molecular characterization of Grp78 revealed it to be a highly conserved Hsp gene, essential not only during infection but also during early developmental stages of rohu, and its expression was noticed in all organs studied except in gill tissues, which indicated its potential immune regulatory role during infection process.     

A 5'-flanking region of embryonic-type myosin heavy chain gene,MYHM743-2, from torafugu Takifugu rubripes regulates developmental muscle-specific expression

The myosin heavy chain gene, MYH_M743-2, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5'-flanking region of MYH_M743-2 into fertilized eggs of zebrafish and medaka. Although the -2.1 kb 5'-flanking region of torafugu MYH_M743-2 showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5'-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1 kb 5'-flanking region of MYH_M743-2 contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka.     

p∆TubHA4C, a new versatile vector for constitutive expression in Drosophila

Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p?TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9 kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ?Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb ΔTub promoter induced similar expression levels to the intact 2.6 kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ?TubHA4C vector.     

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.     

Generation and characterization of a zebrafish muscle specific inducible Cre line

Zebrafish transgenic lines provide valuable insights into gene functions, cell lineages and cell behaviors during development. Spatiotemporal control over transgene expression is a critical need in many experimental approaches, with applications in loss- and gain-of-function expression, ectopic expression and lineage tracing experiments. The Cre/loxP recombination system is a powerful tool to provide this control and the demand for validated Cre and loxP zebrafish transgenics is high. One of the major challenges to widespread application of Cre/loxP technology in zebrafish is comparatively small numbers of established tissue-specific Cre or CreERT2 lines. We used Tol2-mediated transgenesis to generate Tg(CrymCherry;-1.9mylz2:CreERT2) which provides an inducible CreERT2 source driven by muscle-specific mylz2 promoter. The transgenic specifically labels the trunk and tail skeletal muscles. We assessed the temporal responsiveness of the transgenic by screening with a validated loxP reporter transgenic ubi:Switch. Further, we evaluated the recombination efficiency in the transgenic with varying concentrations of 4-OHT, for different induction time periods and at different stages of embryogenesis and observed that higher recombination efficiency is achieved when embryos are induced with 10 μM 4-OHT from 10-somites or 24 hpf till 48 or 72 hpf. The transgenic is an addition to currently available zebrafish transgenesis toolbox and a significant tool to advance muscle biology studies in zebrafish.