The delivery of foreign genes into fertilized fish eggs using high‐velocity microprojectiles

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%, survival rate 25% 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.

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“…This testing is necessary because each species or cell type requires different conditions for DNA transfer (23,24,29,30).…”

Section: Resultsmentioning

confidence: 99%

A biolistic process for in vitro gene transfer into chicken embryos

Ribeiro

Mariani

Azevedo

et al. 2001

Braz J Med Biol Res

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“…Each species or cell type requires different conditions for DNA transfer (Gendreau et al, 1995;Zelenin et al, 1991;Williams et al, 1991), and we have optimized these parameters for chicken embryos.…”

Section: Resultsmentioning

confidence: 99%

In situ DNA transfer to chicken embryos by biolistics

Ribeiro¹,

Azevedo²,

Aragão³

et al. 1999

Genet. Mol. Biol.

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Fertilized chicken eggs were bombarded with a biolistic device. Transient expression of the lacZ gene under the control of a human cytomegalovirus (CMV) promoter was assessed after in situ gene transfer using this approach. The influence of different pressures, vacuum levels and particles was tested. Survival rate improved as particle velocity decreased, but resulted in lower levels of expression. The best survival and expression were obtained with gold particles, a helium gas pressure of 600 psi and a vacuum of 600 mmHg. Under these conditions, all bombarded embryos showed β-galactosidase activity, indicating that this was an effective method for transformation of chicken embryos.

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“…They are relatively more readily available, tractable, small in body size, rapid in development and have short reproductive cycles. They are still popular models because of the establishment of transgenic technologies related to these animals (Fire, 1986;Gordon et al, 1980;Rubin & Spradling, 1982;Zelenin et al, 1991). Such genome manipulation technologies make reverse genetics possible, allowing studies to be amendable for these model organisms.…”

Section: Chilling Injurymentioning

confidence: 99%