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Transfection of DNA to hypertrophic cardiomyopathy with a gene gun

Research Project

Project/Area Number 09557003
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field General anatomy (including Histology/Embryology)
Research InstitutionChiba University

Principal Investigator

TOYOTA Naoji  School of Medicine, Chiba University Lecturer, 医学部, 講師 (00188822)

Co-Investigator(Kenkyū-buntansha) KOMIYAMA Masatoshi  School of Medicine, Chiba University research, 医学部, 助手 (70175339)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
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)
Keywordsgene gun / electroporation / transfection / embryo / heart / pGFP / 筋誘導因子 / in situ hybridization / tag / troponin / C-myc
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 … More 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

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (11 results)

All Other

All Publications (11 results)

  • [Publications] Komiyama M.: "Variations of the extensor indics muscle and tendon"J. Hand Surg. Brit. Eur.. 24B,5. 575-578 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Toyata N.: "Thin filament binding domains of cardiac and fast skeletal muscle troponin I isoforms as studied by epitope tagging"J. Muscle Res. Cell Motil.. 20(8). 755-760 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Komiyama, M. and Toyota, N.: "Variations of extensor in dics muscle and tendon."J. Hand Surg. Brit. Eur.. 24B, 5. 575-578 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Toyota, N.: "Thin filament binding domains of cardiac and fast skeletal muscle troponin I isoform as atudied by epitope tagging."J. Muscle Res. Cell Motil.. 20(8). 755-760 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Komiyama, M.: "Variations of the extensor indics muscle and tendon"J. Hand Surg. Brit. Eur.. 24B, 5. 575-578 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Toyota, N.: "Thin filament binding domains of cardiac and fast skeletal muscle troponin I isoforms as studied by epitope tagging"J. Muscle Res. Cell Motil.. 20(8). 755-760 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Naoji Toyota: "Assembly of force-expressed troponin-I isoforms in myofibrils of cultured cardiac and fast skeletal muscle cells as studied by epitope tagging" J.Muscle Res.Cell Motil.19. 937-947 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hidenori Uzawa: "Force-expression of cardiac troponin I in C2C12 skeletal myoblasts suppresses myoblast fusion and induces actin bundle network formation." Proc.Japan acad.73,Ser.B. 215-218 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Masatoshi Komiyama: "The intracellular sorting of myosin essential light chain isoproteins in chicken fibroblasts." Proc.Japan acad.73,Ser.B. 219-222 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Salma Begum: "Differentiation of muscle-specific proteins in chicken somites as studied by immunofluorescence microscopy." Cell Tissue Res.(in press). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Homare Suzuki: "Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery" Tissue Cell. (in press). (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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