PRODUCT CATEGORY

PRODUCT SEARCH

NEWSLETTER

Register now!Newsletter subscription

  • CATALOG PDF Download
  • BROCHURES PDF Download

Superior to PEG for hybridoma preparation and other cell fusion procedures

GenomONE™ - Neo EX HVJ Envelope Transfection Kit-Technical Support

Technical Support


Troubleshooting Guide for GenomONE - NEO EX

FAQ for GenomONE - NEO EX

 

Troubleshooting Guide for GenomONE-Neo EX

  1. Low transfection efficiency
  2. High cytotoxicity (In vitro)

 

FAQ for GenomONE-Neo EX

  1. Characteristics of HVJ-E
  2. Principle of transfection
  3. Differences in mechanism of transfection between HVJ-E vector and other existing non-viral transfection reagents (cationic liposome etc.)
  4. Restriction for GenomONE-Neo EX use
  5. Expiration date
  6. Particle size of HVJ-E How large is this carrier HVJ envelope vector?
  7. Method in inactivation of HVJ
  8. Assessment and confirmation of viral inactivation
  9. Methods for confirmation of viral inactivation
  10. Bio-safety level for laboratory use
  11. Safety evaluation of HVJ-liposomes in nonhuman primates
  12. Quality assurance
  13. License requirement and commercial use
  14. Role of each Reagent
  15. Constituent and concentration of each Reagent
  16. Storage of reconstituted HVJ-E suspension
  17. Storage of Reagent A, B, C and Buffer
  18. Number of HVJ-E particles included in 1 Assay Unit (AU) of HVJ-E suspension
  19. Hemagglutination units (HAU) of HVJ-E included in 1 Assay Unit (AU) of HVJ-E suspension
  20. Efficiency of incorporation into HVJ-E
  21. Limit of size of DNA that can be incorporated into HVJ-E
  22. Limit of size of protein or synthetic compound  that can be incorporated into HVJ-E
  23. Possibility of storage of HVJ-E vector after incorporation of plasmid DNA, siRNA, ODN or protein.
  24. Recommended cell density for each well plate size
  25. Frequency of use (in vitro)
  26. Effects of serum and antibiotics in the medium during transfection
  27. (In vitro 27. Efficiency of transfection of circular DNA and linear DNA (In vitro)
  28. Transfection efficiency of mRNAs (In vitro)
  29. Frequency of use (in vivo/ laboratory animals)
  30. Precaution for in vivo transfection (route of administration)
  31. Immunogenicity in vivo.Consecutive administration in vivo
  32. Effects of mouse or rat lineage on efficiency of transfection
  33. Published in vivo researches using GenomONE.(Plasmid DNA)

Troubleshooting Guide for GenomONE-Neo EX

Problem

Possible cause

Suggestions and recommendation

Low transfection efficiency

 

Loss of binding or fusion activity of HVJ-E
(In vitro, in vivo)

Check the condition of preparation and storage HVJ-E.

  • Because the activity of freeze-dried HVJ-E can be reduced by exposure to high temperature or high relative humidity, refrigerated storage with sealing in an aluminum bag is required.
  • Freeze-dried HVJ-E should be reconstituted with ice-cooled Buffer and the suspension should be immediately stored in an ice-cooled bath or in a refrigerator (2-8°C). The HVJ-E suspension gradually loses activity if the temperature is above 8°C.
  • After reconstitution, the HVJ-E suspension should be stored in a refrigerator (2-8°C) and should be used within 2 weeks. Since thawing of frozen suspension can reduce transfection activity, reconstituted suspension should not be stored frozen.

Low efficiency of binding of HVJ-E with target cell  membrane
(in vitro)

  • Increase the amount of Reagent C two- to four-fold compared to the standard amount (please note that Reagent C may have cytotoxic effects in some cell lines).
  • Reduce the amount of medium used for transfection and increase the concentrations of HVJ-E vector and Reagent C.
  • (For adherent cells)

Centrifuge the mixture of HVJ-E vector and cells in the plate at 1,500-3,000 rpm for 10-60 minutes at a temperature of 35°C (4°C to room temperature for some types of cells).

  • (For suspension cells)

Extend the duration of centrifugation of the mixture of HVJ-E vector and cells to about 60 minutes.

Low efficiency of incorporation of nucleic acids into HVJ-E
(in vitro, in vivo)

  • Check the incorporation procedure. The amount of Reagent B added should equal to 1/10 of the fluid volume. Excessive addition of Reagent B could result in degradation of membrane function of HVJ-E.
  • Increase the concentration of nucleic acid used for incorporation into HVJ-E two- to four-fold compared to the standard.

Nucleic acids of poor quality
(in vitro, in vivo)

  • Check the purity of nucleic acids. Plasmid DNA used for transfection should be of high quality.
  • Endotoxin level should also be reduced using appropriate purification tools, since endotoxins are reported to be cytotoxic to some cell lines (Huh-7 etc.) and some primary cultured cells.

Cell density is not adequate
(in vitro)

  • Number of target cells may be inadequate. Use adherent cells at 50-80% confluency.

 

Molecular size of content is too small (Mw.<1kD) or too large
(in vitro, in vivo)

  • Using the standard incorporation procedure, molecules with molecular weights less than 1kDa could not be trapped in HVJ-E and might leak through HVJ-E membrane.
  • In contrast, the possibility of incorporating plasmid DNAs or proteins larger than 15kbp (DNA) or 150kDa (IgG), respectively, has yet to be clearly determined.

Serum and antibiotics
(in vitro)

  • Usually, addition of serum and antibiotics to the transfection medium (introduction step) does not affect transfection efficiencies. However, reduction of concentrations of serum or antibiotics might increase transfection efficiency.

High cytotoxicity
(in vitro)

Excessive exposure of cells to HVJ-E vector

  • Reduce the amount of HVJ-E used or the amount of HVJ-E vector added to the medium.
  • (For adherent cells)

Wash the HVE-J vector 10 minutes to 3 hours after addition to the cells, and replace the medium.

  • (For suspension cells)

Shorten the duration of centrifugation after the addition of HVJ-E vector to the cells to the minimum (10 minutes) and set the temperature during centrifugation at 4°C.

Plasmid DNA preparation contaminated with large amount of endotoxin

  • Endotoxin level should also be reduced using appropriate purification tools.

Excessive exposure of cells to Reagent C

  • Reduce the amount of Reagent C (1/2-1/4) or skip use of this reagent.

Conditions of cultured cells are not suitable for transfection

  • Since cells might be contaminated with mycoplasma, check using the appropriate mycoplasma detection tool. Eliminate the mycoplasma or use fresh uncontaminated culture.

If above checks or tests prove negative and do not result in any improvement, HVJ-E vector may be extremely cytotoxic to your specific cell type

  • Since HVJ-E particles strongly bind and fuse with target cell membrane within several minutes, cytotoxicity to very sensitive cell lines may difficult to completely eliminated. However, the following steps can be taken to reduce cytotoxicity.
  • In the introduction step, binding of HVJ-E vector and cells is performed at 4°C or on ice for 15 min (binding of HVJ-E particles to cell membrane is achieved at even lower temperature), remove the HVJ-E vector thereafter and replace the medium.
  • Perform the transfection at a higher cell density.

FAQ for GenomONE-Neo EX

 

Question

Answer and comment

1

Characteristics of HVJ-E

GenomONE-Neo EX is a non-viral transfection reagent. Since genomic RNA of which has been completely inactivated, it has neither infective nor proliferative potentials in humans or animals.

2

Principle of transfection

The molecule to be transferred (DNA, protein, antisense oligonucleotide, siRNA, etc.) is incorporated in HVJ-E particles to yield an HVJ-E vector, which is then introduced into the target cell or tissue, making use of the sialic acid-binding activity of HN protein and membrane-fusion activity of F (fusion) protein existing in HVJ-E envelope.

3

Differences in mechanism of transfection between HVJ-E vector and other existing non-viral transfection reagents (cationic liposome etc.)

Conventional non-viral transfection reagents, including cationic lipids are incorporated into cells through endocytosis which results in degradation of most regions of the transferred DNAs or other molecules by lysosomes.
  Unlike these vector systems, HVJ-E vector resists degradation by lysosomes, making it easy to transfer the specified molecules directly into the cytoplasm. Therefore, the HVJ-E vector thus yields highly efficient transfection.

4

Restriction for GenomONE-Neo EX use

GenomONE is developed, designed and sold for research purposes only. It is not to be used for human or animal diagnostic or therapy (drug purposes).

5

Expiration date

The period of guarantee of quality for freeze-dried HVJ-E is printed on the aluminum pack of HVJ-E.

6

Particle size of HVJ-E
How large is this carrier HVJ envelope vector?

The average diameter of each HVJ-E particle is approximately 300 nm (200-400 nm).

7

Method in inactivation of HVJ

Although HVJ-E uses HVJ (Sendai virus/ Murine parainfluenza virus 1) as a raw material, the genomic RNA of HVJ has been completely inactivated by drug treatment*.  The HVJ-E will not proliferate or exhibit pathogenic effects in humans or animals.
* Reference:
Prior, P. et al.: BioPharm, 22-33 (Oct. 1996)
Kaneda, Y. et al.: Advances in Genetics, Vol. 53, pp308-332 (2005).

8

Assessment and confirmation of viral inactivation

Inactivation of HVJ has been confirmed for each lot by the viral proliferative potential rule-out test, using cultured cells and growing chicken eggs.

9

Methods for confirmation of viral inactivation

Lack of possibility of infection or proliferation of HVJ-E in humans or experimental animals has been confirmed by means of the following three methods.

  • Assay using cultured cells
  • Assay using fertilized chicken eggs
  • Assay using mice

10

Bio-safety level for laboratory use

GenomONE can be used safely in ordinary laboratories.
Although HVJ-E uses HVJ (Sendai virus/Murine parainfluenza virus 1) as a raw material, the genomic RNA of HVJ has been completely inactivated by drug treatment. HVJ-E will not proliferate or exhibit pathogenic effects in humans or animals.
However, when using this product for recombinant DNA experiments, rules for recombinant DNA experiments (stipulated in relevant statutes in the country of use or set forth by the safety committee of the facility concerned) must be followed, and experiments should only be carried out in laboratories properly equipped with facilities appropriate for recombinant DNA experiments.

11

Safety evaluation of HVJ-liposomes in nonhuman primates

Safety of the HVJ-liposome vector (a hybrid vector of HVJ-E and liposomes) in cynomolgus monkeys has been demonstrated in the following paper.
Tsuboniwa et al,: Human Gene Therapy, 12, 469-487 (2001).

12

Quality assurance

  • Inactivation of HVJ has been confirmed for each lot by the viral proliferative potential rule-out test, using cultured cells and fertilized chicken eggs. (Details, see the next column)
  • Absence of contamination by bacteria and fungi has been confirmed by sterility testing.
  • Endotoxin level has been confirmed to be less than 2.5 EU/mL (Limulus Amebocyte lysate gel clot assay).
  • Expression of the gene introduced in cultured cells (BHK-21; ATCC CCL-10) in the presence of serum has been confirmed.

13

License requirement and commercial use

This product and its use are covered by the claims of one or more patents (including patents pending) and licensed for research use only. It may not be used for any commercial or other purpose or resold after modification or the like without prior written approval from manufacturer (Ishihara Sangyo Kaisha, Ltd.).

14

Role of each Reagent

  • 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.
  • 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.
  • Reagent B: Increases permeability across the HVJ-E membrane.
  • 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.
  • Buffer: Neutral buffer of physiological concentration used for suspending or diluting HVJ-E or other purposes.

15

Constituent and concentration of each Reagent

Not disclosed.

16

Storage of reconstituted HVJ-E suspension

Do not freeze. The reconstituted HVJ-E suspension should be stored in a refrigerator (2-8°C) and should be used within 2 weeks. Since thawing of frozen suspension can reduce activity, The suspension should not be stored frozen.

17

Storage of Reagent A, B, C and Buffer

Do not freeze. Stored in a refrigerator (2-8°C).

18

Number of HVJ-E particles included in 1 Assay Unit (AU) of HVJ-E suspension

1 Assay Unit (AU) [40mL of reconstituted HVJ-E suspension] includes approximately 109-1010particles of HVJ-E.

19

Hemagglutination units (HAU) of HVJ-E included in 1 Assay Unit (AU) of HVJ-E suspension

1 Assay Unit (AU) [40mL of reconstituted HVJ-E suspension] is equivalent to approximately 1,000-2,000 hemagglutination unit (HAU).

20

Efficiency of incorporation into HVJ-E

The efficiency of incorporation plasmid DNA is estimated to be approximately 15-20%. Kaneda et al., Mol. Ther.: 6, 219-225 (2002)

21

Limit of size of DNA that can be incorporated into HVJ-E

Plasmid DNAs of 10 to 15 kbp in size are successfully incorporated into HVJ-E particles and introduced to target cells. Limit of the molecular size to be incorporated into the HVJ-E has yet to be clearly demonstrated.

22

Limit of size of protein or synthetic compound  that can be incorporated into HVJ-E

Fluorescence-labeled proteins with their molecular weight of 7kDa (insulin) to 150kDa (rabbit IgG) are successfully incorporated into HVJ-E particles.
Synthetic compounds (molecular weight >1,000) can also be incorporated into HVJ-E.

23

Possibility of storage of HVJ-E vector after incorporation of  plasmid DNA, siRNA, ODN or protein.

HVJ-E vector (with specified molecule incorporated) should be used within the day of preparation.

24

Recommended cell density for each well plate size

Adherent cells

Plate

Cell density (upon inoculation onto the well plate*)

6-well plate

0.4 - 2.0 ´ 105 cells/2.0 mL of medium/well

24-well plate

1.0〜5.0×104 cells/0.5mL of medium/well

96-well plate

0.25〜1.25×104 cells/0.125mL of medium/well

* Used for transfection under conditions of one-day culture and 50-80% confluency.

Suspension cells

When transfection of suspension cells is performed, the cells are combined with HVJ-E vector in a tube, and the mixture is centrifuged to induce contact between the cells and the vector, leading to transfection.


Plate

Cell density

Centrifugation
(in a tube)

Medium for resuspension

Inoculation onto the well plate

6-well

0.4〜2.0×106 cells/0.5mL of medium/tube

2.0mL

0.4〜2.0×106 cells/2.0mL of medium/well

24-well

0.2〜1.0×106 cells/0.25mL of medium/tube

1.0mL

1.0〜5.0×105 cells/0.5mL of medium/well

96-well

0.25〜1.25×105
cells/0.125mL of
medium/well


Transfection of suspension cells

25

Frequency of use
(in vitro)

If used with the method described in this package insert, the product (GN004EX)  be used for 25 assays (with a 6-well plate or a 35mm dish).  If used for siRNA oligo, it can be used for 100-200 assays (with a 6-well plate).

26

Effects of serum and antibiotics in the medium during transfection (In vitro)

Usually, addition of serum and antibiotics to the transfection medium (introduction step) does not affect transfection efficiencies.

27

Efficiency of transfection of circular DNA and linear DNA (In vitro)

With regard to HVJ-E system, there are no clear evidence that transfection efficiency of linear DNA is higher than that of circular DNA.

28

Transfection efficiency of mRNAs (In vitro)

For the HVJ-E system, there is no clear evidence that transfection efficiency of mRNA is higher than that of plasmid DNA.

29

Frequency of use
(in vivo/ laboratory animals)

The amount of HVJ-E required is approximately 1-2 AU (40 to 80 μL of reconstituted HVJ-E suspension) for mice and 5-10 AU (200 to 400 μL) for rats. Therefore, the product (GN004EX; 0.26mLx 4 vials/kit) can be used for 12-25 mice and 2-5 rats.
However, since optimal conditions of transfection in vivo can vary markedly depending on the route of administration and the type or location of the target organ, it is advisable to optimize the dose level in each experiment.

30

Precaution for in vivo transfection
(route of administration)

Because HVJ-E can be adsorbed onto tissue in vivo, especially blood cells through the HN envelope protein, the efficiency of transfection in vivo through intravenous (systemic) administration is usually very low. It is advisable to select a route of administration involving less exposure to blood or to perform perfusion of the animal prior to administration.
However, transfections through intravenous injection into mouse has been reported in the following articles.
1) Matsuda N., et al.; Nuclear factor κ-B decoy oligodeoxynucleotides prevent acute lung injury in mice with cecal ligation and puncture-induced sepsis. 
Mol. Pharmacol., 67 (4), 1018-1025 (2005).
2) Takeno M. al. : Th1-dominant shift of T cell cytokine production, and subsequent reduction ofserum immunoglobulin E response by administration in vivo of plasmid expressing Txk/Rlk, a member of Tec family tyrosine kinase, in a mouse model.
Clin. Exp. Allergy, 34, 965-970 (2004).
3) Kaneda Y. et al.; Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system.
Mol.Ther., 6 (2), 219-226 (2002).

31

Immunogenicity in vivo.
Consecutive administration in vivo

Antibodies to HVJ-E components (e.g. F or HN envelope protein) will be produced after injection of the vector. However, Kaneda et al. have shown that luciferase gene expression is not inhibited after consecutive administration of HVJ-E into mouse muscle (twice in a 2-week interval).
Reference; Kaneda, Y. et al.: Advances in Genetics, 53, 308-332 (2005).

32

Effects of mouse or rat lineage on efficiency of transfection

The type of mouse or rat lineage is not a critical factor affecting efficiency of gene expression. Other factors, including a promoter systems or purity of DNA preparation might have critical effects on this efficiency.

33

Published in vivo researches using GenomONE.
(Plasmid DNA)

Product List

Product Name Cat# Quantity Price

GenomONE-Neo EX HVJ-E 1 vial Transfection Reagents

ISK-GN-001-EX 1SET

¥ 25,000
$ 334
€ 250

GenomONE-Neo EX HVJ-E 4 vials Transfection Reagents

ISK-GN-004-EX 1SET

¥ 77,000
$ 1027
€ 770