<siRNA Transfection> 

 Comparison of activity of introduced siRNA between GenomONE-Neo and
 liposomal reagents

【Example1】 
     Suppression of cell proliferation by Eg5 knock down 
 

  Eg5-siRNA was incorporated in each reagent to a final concentration of 50 nM in a 96-well plate. 
  Cells of various lines were treated with the reagents. Forty-eight hours later, the percentage of 
  viable cells was measured by WST-1 assay. Eg5 knock-down resulted in suppression of 
  cell proliferation and induced apoptosis. The finding of a lower percentage of viable cells 
  indicated stronger Eg5 knock-down effects. 

 【Example 2】
     Introduction of Bin-1 siRNA(C2C12)
    

  【Data】 Dr. Chie Kojima* and Dr. Hisataka Sabe 
             Department of Molecular Biology, Osaka Bioscience Institute, Japan. 
           * Department of Applied Chemistry, Graduate School of Engineering, 
             Osaka Prefecture University, Japan.
  【Related article】 Kojima C, et al.:　EMBO Journal, 23, 4413-4422 (2004).　　 

 【Example 3】  
      Phospholamban (PLB) knock-down in primary rat myocardial cell cultures
     

	GenomONE-Neo                              Cationic Lipid reagent
	The efficiency of siRNA transfer into cells was high (80-100%) with both reagents. However, compared to cationic lipid, GenomONE-Neo  induced knock-down of the target protein more efficiently at lower concentrations of siRNA.

    
  【Data】 Dr. Masashi Arai and Dr. Atai Watanabe
         　  Department of Medicine and Biological Science, Gunma University 
             Graduate School of Medicine, Japan. 
  【Related article】  Watanabe A, et al.: J. Mol. Cell. Cardiol., 37, 691-698 (2004).

 <Protein(Enzyme) transfection> 
    
 Comparison of activity of introduced protein between GenomONE  and
 other protein delivery reagents
    
 【Example 1】  
      Tumor cell death induced by intracellular delivery of RNase T1 (SAS cells)

  RNaseT1 was delivered into SAS cells (tongue derived squamous cell carcinoma) using GenomONE (HVJ-E) 
  or two alternative protein delivery reagents. Twenty hours later, cellular metabolic activity (cytotoxicity) was 
  assessed by means of a WST-1 assay.
  RNaseT1 alone did not induce tumor cell death because of its inability to permeate the cell membrane (data not shown).
  In contrast, RNaseT1 incorporated in HVJ-E induced cytotoxocity in a concentration dependent manner, suggesting that 
  intact enzyme was delivered into the cytoplasm without losing its activity. Cytotoxic activity induced by RNaseT1/HVJ-E 
  was higher than those induced by the other two reagents.
    
  【Related article】 Yuki S. et al.:　Eur. J. Biochem., 271, 3567-3572(2004).
   

 【Example 2】
       Delivery of ß-Galactosidase into NIH-3T3 cells
  HVJ-E vector system bypasses degradation or denaturation by lysosomal enzymes, making it easy to uniformly deliver 
  the bioactive proteins into the cytoplasm

  β-Gal was delivered into cells using each reagent. After incubation for four hours, non-specifically bound β-Gal was 
  degraded by trypsin treatment. Cells were then treated with X-Gal reagent to detect β-Gal-expressing cells.
  Uniform distribution of β-Gal was observed when GenomONE (HVJ-E) was used, whereas delivery of protein using
  the other two reagents was not uniform. Aggregation of delivered protein in the cells was apparent 
  when Product B was used.

        PAGE analysis (non-denaturing condition) of ß-Gal protein extracted from
        cells （X-Gal staining）

 　Four hours after intracellular delivery of β-Gal, cells were collected and lysed by freezing and thawing, and then analyzed
　 by  PAGE under non-denaturing conditions (without SDS) followed by staining with X-Gal reagent. 
　 X-Gal stained-positive clear band （Lane 3） with the same molecular weight as standard β-Gal （Lane 6, 7） was detected
   when GenomONE （HVJ-E） was used. This result suggests that β-Gal incorporated in the HVJ-E was delivered into  
   cytoplasm without degradation.In contrast, β-Gal molecules extracted from cells in which cationic lipid-based two other
   reagents were used for delivery exhibited smeared patterns （Product A; Lane 1, Product B; Lane 2）, suggesting that 
   the molecules could be degraded during the introduction step.Unlike other lipid-based reagents, HVJ-E delivers the
   specified proteins directly into the cytoplasm through membrane fusion. Therefore, the HVJ-E system has an advantage
    that it resists degradation by lysosomal enzymes.

  Simultaneous Expression of Two Different Plasmids

   Quantitative and simultaneous expression both the luciferase gene (pGL3) and the alkaline phosphatase gene (pSEAP2) in 
   BHK-21 cells was achieved, in which interference by either gene was not apparent. 

 【Example 1】
       Amount of pGL3 (luciferase gene) plasmid was varied 
      Serial two-fold dilution samples of pGL3 plasmid （860～1.7 ng/well） were mixed with equal amounts of pSEAP2 
      plasmid （860 ng/well）. After incorporation into HVJ-E, they were introduced into BHK-21 cells.

  【Example 2】
      Amount of pSEAP2 (alkaline phosphatase gene) plasmid was varied
      Similar results were obtained when the amount of pSEAP2 plasmid was varied instead（860～1.7 ng/well).

   High-Throughput Screening (HTS) of Functional Genes Using an HVJ-Envelope Vector