| Budget Amount *help |
¥10,500,000 (Direct Cost: ¥10,500,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1997: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Research Abstract |
We tried to introduce DNA to cardiac muscle by electroporation instead of a gene gun. The gene gun is disadvantageous for transfect embryonic heart, because it is too soft to accept shot gold particles covered with DNA.
By using in ovo electroporation (EP), plasmid DNAs (pmiwZ and pGFP) were transfected to living chicken embryos at 3 days incubation. The pmiwZ is a reporter gene containing the bacterial lacZ gene driven by the RSV-LTR and chicken β-actin promoters. The pGFP is a vector carrying green fluorescent protein (GFP) drove by the CMV promoter. The blunt edge of the fertilized eggs incubated for 48-72 hrs were cleaned with 70% ethanol, and a hole of approximately 20 mm in diameter was made on the egg shell. Shell membrane was carefully removed to visualize the embryo. Through the hole, the DNA solution containing 5-10 μg of plasmids dissolved in 10 μl of TE buffer (10 mM Tris-HCl, pH 7.5 and 1 mM EDTA) was dropped on the heart. L-shaped electrodes 5 mm long were placed in the ar
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ea opaca along with the embryonic body on both the right and left sides at 10 mm interval so as to cover the heart region. Effects of varying voltages and time constants were examined to obtain optimal conditions for in ovo EP with an electro-square porator T820 (BTX, San Diego, USA). After the DNA transfection, the hole was sealed with a mending tape. At 3 days after transfection, expression of lacZ and green fluorescence were detected by conventional and fluorescence microscope, respectively. The highest gene transfection efficiency was obtained by applying 8 times of electric square pulses at 25 V in combination with 50 msec. The population of surviving embryos was approximately 78% (N=57). The proportion of the gene positive embryos with surviving ones were approximately 37%. No significant difference was found in pmiwZ and pGFP. The transfected area was approximately 2-3 mm in diameter and 1-2 mm in depth from surface of embryos. In limb buds, heads, and tails of embryos, lacZ and GFP were positively expressed effectively, but in heart, no expression of the genes was found. It suggests that hart was hardly transfected by EP. The use of adenovitus or retrovirus integrates may be advantageous to introduce DNA to heart. Less
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