Temporal expression pattern of myostatin transcripts during chicken embryogenesis

No presente estudo, estimou-se a abundância dos transcritos da miostatina foi estimada durante a embriogênese de galinha por análises de RT-PCR competitiva. Níveis basais de mRNA desse gene foram detectados até o estádio HH15, enquanto acúmulos significativos nesses níveis foram observados apenas no estádio HH24, seguido por redução na abundância desses transcritos a partir do estádio HH26. Tais descobertas preliminares proporcionam informações relevantes sobre a ativação do fator de crescimento miostatina durante o desenvolvimento in ovo de aves.

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping á-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19 percent was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Influence of the neural tube/notochord complex on MyoD expression and cellular proliferation in chicken embryos

Important advances have been made in understanding the genetic processes that control skeletal muscle formation. Studies conducted on quails detected a delay in the myogenic program of animals selected for high growth rates. These studies have led to the hypothesis that a delay in myogenesis would allow somitic cells to proliferate longer and consequently increase the number of embryonic myoblasts. To test this hypothesis, recently segmented somites and part of the unsegmented paraxial mesoderm were separated from the neural tube/notochord complex in HH12 chicken embryos. In situ hybridization and competitive RT-PCR revealed that MyoD transcripts, which are responsible for myoblast determination, were absent in somites separated from neural tube/notochord (1.06 and 0.06 10-3 attomol MyoD/1 attomol á-actin for control and separated somites, respectively; P<0.01). However, reapproximation of these structures allowed MyoD to be expressed in somites. Cellular proliferation was analyzed by immunohistochemical detection of incorporated BrdU, a thymidine analogue. A smaller but not significant (P = 0.27) number of proliferating cells was observed in somites that had been separated from neural tube/notochord (27 and 18 for control and separated somites, respectively). These results confirm the influence of the axial structures on MyoD activation but do not support the hypothesis that in the absence of MyoD transcripts the cellular proliferation would be maintained for a longer period of time

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100 percent, survival rate 25 percent and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters

Selection for breed-specific growth hormone and IGF-I alleles in a synthetic beef cattle cross, Canchim

Este estudo foi desenvolvido com o objetivo de avaliar as freqüências gênicas em diferentes geraçöes de bovinos da raça sintética Canchim (5/8 Charolês, 3/8 Zebu). Uma amostra de 154 animais, representando três classes de geraçöes de um rebanho da raça Canchim, foi analisada para sete marcadores moleculares. Uma amostra da raça Charolesa (N=36) foi incluída nas análises para permitir comparaçöes. Observou-se um aumento linear, altamente significativo (P < 0,01), na freqüência do alelo que codifica o aminoácido valina na posiçäo 127 do hormônio de crescimento, ao longo das geraçöes de Canchim. Este alelo foi observado na amostra da raça Charolesa e näo é encontrado em raças zebuínas nacionais. Quatro alelos foram observados para o microssatélite localizado na regiäo 5' näo transcrita do gene do fator de crescimento semelhante à insulina do tipo I (IGF-I), com tamanhos variando de 231 a 225 pb. A freqüência do alelo de 225 pb apresentou um aumento significativo (P < 0,05) näo linear ao longo das geraçöes. Este alelo näo foi observado na raça Charolesa e é predominante nas raças zebuínas. Para o microssatélite CSFM50, seis alelos foram observados na raça Canchim, tendo sido verificada uma reduçäo linear (P < 0,05) na freqüência do alelo de 168 bp. A hipótese de acasalamento preferencial com relaçäo ao polimorfismo do hormônio de crescimento foi reforçada pelos resultados da análise de estatística F de Wright. O valor estimado de Fis para este loco foi 0,59 (P < 0,01). O aumento simultâneo de um alelo do hormônio de crescimento característico de Bos taurus e de um alelo de IGF-I característico de Bos indicus sugere que a seleçäo fenotípica tenha favorecido regiöes diferentes de ambos os genomas que entraram na formaçäo dessa raça.