Transferência gênica mediada por espermatozóides para geração de jundiás (Rhamdia quelen) transgênicos / Sperm Mediated Gene Transfer for generation of transgenic jundiás (Rhamdia quelen)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Various methodologies to insert exogenous DNA into eukaryotic cells have been studied in the last two decades. In fish, the technique most studied to the moment is microinjection, which promotes the insertion of fragments of interest genes into zygotes. However, this methodology has some negative aspects: it is time and work consuming, requires qualified labor force, the embryo mortality is high, the percentage of integration is frequently low, the opacity of the fish egg does not allow the visualization of the pro-nuclei and the chorionic layer of various species of fish is very resistant, consisting a barrier to microinjection. An alternative to improve the process of exogenous DNA injection would be the mass transfection of spermatic cells in vitro before fertilization. Sperm-mediated gene transfer (SMGT) has been used to study and to generate transgenic animals in different species, including fish. Some tools like electroporation, osmotic potential, and sperm incubation are alternatives that could improve the SMGT in fish. This study aimed to evaluate the potential of jundiá (Rhamdia quelen) sperm, submitted to different treatments of exogenous DNA insertion, to generate transgenic jundiás. To reach this objective, two linear vectors, pCMV-GFP and pcBA-GFP, were used in five treatments: dehydration/rehydration (DR), electroporation (E), dehydration/rehydration/electroporation (DRE), incubation with seminal plasma (INC), and incubation without seminal plasma (INCSP). As control treatment, sperm diluted in isotonic solution without exogenous DNA was used. To evaluate sperm potential, spermatic activity (AT), duration of spermatic activity (TPA), membrane integrity, and in vitro fertilization (FIV) through FIV percent (TXFIV) and hatching percent (TXECLO) were measured. To evaluate GFP expression, animals were anesthetized and observed under fluorescent microscope. Statistical analyses showed a significant effect (P <0.05) of the treatments in AT, TPA, TXFIV, and TXECLO. It was observed that TXFIV has a significant correlation (P <0.01) with AT in all treatments. DRE treatment showed mean spermatic activity significantly superior to control, and it was also the treatment that generated the higher percentage of animals with transient expression of GFP (63%). All treatments were able to promote jundiá sperm transfection and to generate animals with different degrees of transient GFP expression. GFP expression patterns were observed into muscular and nervous tissues, reflecting the promoters used, and demonstrating that both vectors were carted by the sperm cells. This study demonstrated the high biological potential of jundiá sperm cells to genic manipulation and that it is possible to mass generate transgenic jundiás.

ASSUNTO(S)

rhamdia quelen transferência genética mediada por espermatozóides jundiá dna exógeno smgt biotecnologia animais transgênicos transfeccção celular genetica animal

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