For Laboratory Research Use Only. Not for Use in Clinical Diagnosis or Therapeutic Procedures.

HVJ-E Transfection Kit

 Product Name  Cat # Size

 Price

GenomONE-Neo EX     GN001EX 1kit (HVJ-E 1 vial set) distributor website
GN004EX 1kit (HVJ-E 4 vials set) distributor website
GN040EX 1kit (HVJ-E 40 vials set) distributor website


  GenomONE-Neo EX  (HVJ Envelope VECTOR KIT) is a novel and unique transfection kit

  which employs the membrane fusion ability of the envelope of Sendai virus
  (Hemagglutinating virus of Japan: HVJ).

    
   
What's HVJ Envelope (HVJ-E)?
       

 [Features] 

  1: Wide usability
      You can use our product for transfection from in vitro to in vivo.

 
2: Introduction of various molecules
      You can incorporate not only plasmid DNA but also other molecules such as siRNA, proteins,  
      anti-sense oligonucleotides into HVJ Envelope. You can also incorporate multiple species of molecules
 
      simultaneously.

 
3: Safe and easy
      The genome RNA of HVJ is completely inactivated, so that you can use it as a "non-viral" transfection reagent 
      safely and easily.
   

 [Principle] 

Conventional non-viral transfection tools, including cationic lipids, are incorporated into cells through endocytosis which results in degradation of most parts of the transferred DNA by lysosomes. On the other hand, HVJ Envelope VECTOR resists degradation by lysosomes, making it easy to transfer the specified  DNA. Therefore, HVJ Envelope VECTOR yields highly efficient gene expression. Sialic acid receptors, which are needed to trigger binding with HN protein, exist in almost all animal cells. Thus, HVJ Envelope VECTOR is useful for a wide range of targets.


    


 

 

  [Applications]                                                                                  

                         

<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. 

    
  【
DataDr. 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 -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

  Delivery of -Gal protein Phase contrast

   GenomONE                                      Product A                                      Product B

 

  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 -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.

 

 <Plasmid DNA transfection> 

 
Application for High-Throughput Screening of Functional Genes

  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
8601.7 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 instead8601.7 ng/well).

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

   Advantages of an HTS system based on the HVJ-E vector
   
n Rapid preparation of the vector containing the DNA library
  
n Effective membrane fusion-mediated introduction of the DNAs into various cells
   n Easy cloning of candidate genes by transformation of E.coli.
  
n Minimization of the time needed to screen for functional genes

  【Related Articles
  Nishikawa T. et al.: Development of high-throughput functional screening of therapeutic genes, 
  using a hemagglutinating virus of Japan envelope vector. 
  Hum. Gene Ther., 17(4), 470-475 (2006).

  Takami Y. et al.: Ubiquitin carboxy-terminal hydrolase L1, a novel deubiquitinating enzyme in the vasculature, 
  attenuates NF-kB activation.  
  Arterioscler. Thromb. Vasc. Biol., 27, 2184-2190 (2007).
  


 [Specifications] 

     Cat. #

   Freeze-dried HVJ-E
      (inactivated HVJ)  

           0.26 mL /vial

     (when reconstituted)

           Reagent A 
 
(enhancer for incorporation)

            
0.5 mL/vial  

          Reagent B        
 
(reagent for incorporation)

          
0.3 mL/vial

          Reagent C   
 (enhancer for introduction)

           
1.0 mL/vial 

ISK-GN001EX  

             1 vial

                 1 vial  

                1 vial

               1 vial

ISK-GN004EX  

             4 vials

                 1 vial

              1 vial

             1 vial

ISK-GN040EX  

              40 vials

               10 vials

              10 vials

             10 vials

 

 [Storage] 

 n  [Freeze-dried HVJ-E]: Refrigerated at 2-8C, sealed in an aluminum package.  Keep dry.

 n  [Reagent A, B, C and Buffer]: Refrigerated at 2-8C.

 n  [Reconstituted HVJ-E suspension]: Refrigerated at 2-8C and should be used within 2 weeks.
                                                          Do not freeze.

 [Role of each reagent] 
  n  [Freeze-dried HVJ-E]: The main frame of the vector into which the molecule to be transferred is included.
                                                 It fuses with the cell membrane, allowing the target molecule to be introduced into
                                      the cytoplasm.
 
n  [Reagent A]: A positively-charged peptide which increases the affinity between the target molecule and 
                              HVJ-E and thus facilitates incorporation of the molecule into HVJ-E.
  n [Reagent B]: Increases permeability across the HVJ-E membrane.
  n [Reagent C]: A positively-charged peptide which increases affinity between the molecule-bearing HVJ-E
                         (HVJ-E vector) and the cell (or tissue) and thus increases the efficiency of transfection.
 
n [Buffer]: Neutral buffer of physiological concentration used for suspending or diluting HVJ-E or other purposes.
  

 [Frequency of use] 
  n  If used with the method described in this package insert, the product can be used for transfection 

     as follows (with a 6-well plate).

      Cat. #

No. of HVJ-E vials per kit

                            Cargo to be transfected

  Plasmid DNA, ODN, protein

            siRNA (oligo-type)

  ISK-GN001EX

                1

             6 assays (wells)

             25-50 assays (wells)

  ISK-GN004EX

                4

           25 assays (wells)

          100-200 assays (wells)

  ISK-GN040EX

               40

         250 assays (wells)

     1,000-2,000 assays (wels)

   
 [Related Technical Information] 
  

  

 

[Related product] 

Antibody delivery reagent 

 (For introduction of antibody)

GenomONE-CAb EX 

Cell fusion kit

 [For preparation of hybridomas (monoclonal antibodies) and

  research on development, differentiation, and breeding

  (transplantation and replacement of nuclei)]

GenomONE-CF EX 

 


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  siRNA data sheet

 



Data Sheet for 
Protein Delivery

 



Reference list
(in vitro)

 



Reference list
(in vivo)

 



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