• Tiago Collares

      Articles written in Journal of Biosciences

    • Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer

      Tiago Collares Vinicius Farias Campos Fabiana Kömmling Seixas Paulo V Cavalcanti Odir A Dellagostin Heden Luiz M Moreira João Carlos Deschamps

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      The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E, INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups (𝑃 > 0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology

    • Identification, tissue distribution and evaluation of brain neuropeptide Y gene expression in the Brazilian flounder Paralichthys orbignyanus

      Vinicius F Campos Tiago Collares João C Deschamps Fabiana K Seixas Odir A Dellagostin Carlos Frederico C Lanes Juliana Sandrini Luis Fernando Marins Marcelo Okamoto Luís A Sampaio Ricardo B Robaldo

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      Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 ± 3°C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian flounder are affected by temperature.

    • Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or 𝑁,𝑁-dimethylacetamide

      Tiago Collares Vinicius Farias Campos Priscila Marques Moura De Leon Paulo V Cavalcanti Marta G Amaral Odir A Dellagostin João Carlos Deschamps Fabiana K Seixas

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      Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or 𝑁,𝑁-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA–DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.

    • TP53 gene polymorphism: Importance to cancer, ethnicity and birth weight in a Brazilian cohort

      Helena S Thurow Ricardo Haack Fernando P Hartwig Isabel Ode Oliveira Odir A Dellagostin Denise P Gigante Bernardo L Horta Tiago Collares Fabiana K Seixas

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      Arg72Pro SNP of p53 has been associated with many types of cancer as well as with survival and longevity. We evaluated the Arg72Pro SNP frequencies of a Brazilian birth cohort and their association with current, demographic and birth epidemiological parameters available. In 1982, all hospital births of Pelotas, southern Brazil, were identified and studied prospectively. In 2004–5, blood samples were collected and DNA extracted. PCR-RFLP was used to genotype the Arg72Pro SNP in 3794 individual samples of the Brazil birth cohort and DNA sequencing was performed to confirm the genotypes. The genotype distribution, which was in Hardy–Weinberg equilibrium, showed a predominance of the arginine amino acid with a frequency of 46.9% Arg/Arg, 42.2% Arg/Pro and 10.9% Pro/Pro. The allele frequency was 0.68 of Arginine and 0.32 of Proline. The Arg72Pro SNP genotype and allelic frequency were related to skin colour where proline amino acid was observed more among black subjects, while arginine amino acid was observed more among white subjects. The individuals without family history of cancer and those with low birth weight were associated with arginine amino acid. The Arg72Pro SNP was strongly associated with important epidemiological variables confirming that genetic profiles on cohort studies can improve our understanding of the susceptibility of diseases and its risk factors.

    • Neuropeptide Y gene expression around meal time in the Brazilian flounder Paralichthys orbignyanus

      Vinicius F Campos Ricardo B Robaldo João C Deschamps Fabiana K Seixas Alan John A McBride Luis Fernando Marins Marcelo Okamoto Luís A Sampaio Tiago Collares

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      Neuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (−2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (𝑃 < 0.05) and significantly reduced 2 h later (𝑃< 0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (𝑃 < 0.05) at −2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.

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