®

GenomONE series

HVJ Envelope transfection and cell fusion kits

ISHIHARA SANGYO KAISHA, LTD.

https://www.iskweb.co.jp/eng/products/hvj-e/

Genome EditingMembrane fusion-mediated intracellular delivery of Cas9 protein enables

efficient genome editing even in mouse primary T cells.

・Applicable to CRISPR-Cas9-mediated knock-in and knock-out

・Higher genome editing efficiency than that of the competitors

TransfectionHighly efficient transfection of plasmid DNA, siRNA/miRNA, and protein

・Applicable to immune cells (e.g., Jurkat, THP-1, U-937, RAW 264.7, and mouse primary T cells)

・Applicable to in vivo transfection

Cell fusionAlternative method to PEG and electrofusion for cell fusion

・Simple operability, low cytotoxicity and high fusion activity

・Applicable to hybridoma preparation and nuclear transfer

GenomONE series

GenomONE-NeoPlasmid DNA, siRNA/miRNA, protein transfection kit (in vivo)

p. 4GenomONE-SisiRNA/miRNA transfection kit

p. 5

GenomONE-CFCell fusion kit（hybridoma preparation, nuclear transfer）

p. 7

GenomONE-GECas9 protein and gRNA transfection kit

p. 3

GenomONE-GXPlasmid DNA transfection kit (in vitro)

p. 6

■■■■ Table of contents

■■■■ What’s HVJ Envelope?

HVJ Envelope(HVJ-E) is a novel and unique vesicle that employs the membrane fusion

ability of the envelope of Sendai virus (Hemagglutinating virus of Japan; HVJ). The

genome RNA of HVJ is completely inactivated to use HVJ-E in biosafety level 1(BSL1)

laboratories.

HVJ-E is known for its ability to incorporate a variety of molecules, such as plasmid

DNA, siRNA/miRNA or proteins and introduce them into the cytoplasm via membrane

fusion. HVJ-E can also be used as a cell fusion reagent.

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One day after stimulation of mouse splenocytes with PMA/ionomycin, T cells were isolated. Cas9 protein and PPIB-targeting gRNA

were then transfected using GenomONE-GE, product C, or product T (competitors). Two days after transfection, genome editing

efficiency was evaluated using T7 endonuclease I mismatch cleavage assay.

GenomONE - GE・Optimal for transfection of Cas9 protein and gRNA

・Applicable to transfection of Cas9 protein, gRNA and donor DNA (knock-in)

・Applicable to difficult-to-transfect cells (e.g., U-937, Jurkat, and mouse primary T cells)

■■■■ Cas9 protein and PPIB-targeting gRNA transfection in HeLa and U-937 cells

Cas9 protein and PPIB-targeting gRNA were transfected into HeLa and U-937 cells using GenomONE-GE, product C, or product T

(competitors). Two days after transfection, genome editing efficiency was evaluated using T7 endonuclease I mismatch cleavage

assay.

3

®

HeLaU-937

Cleaved efficiency (%) = sum of cleaved band intensities/(sum of the cleaved and parental band intensities) x100

■■■■ Cas9 protein and PPIB-targeting gRNA transfection in mouse primary T cell

■■■■ Cas9 protein, gRNA, donor DNA transfection (knock-in)

To introduce the BamHI restriction site into the

PPIB gene, Cas9 protein, PPIB-targeting gRNA,

and ssDNA containing BamHI restriction site

were transfected using GenomONE-GE. Two days

after transfection, knock-in efficiency was

evaluated using RFLP.

A knock-in efficiency of 14% was obtained using

GenomONE-GE.

0

0.2

0.4

0.6

0.8

1

1.2

Control(PBS) Product X Product R GenomONE-si

HL-60 Negative control siRNA

CDC2 siRNA

As a kinesin-like motor protein, Eg5 (KIF 11) is essential for the

formation of spindle microtubules during cell division, and when

its function is inhibited, cell division is stopped and apoptosis is

induced. Based on such a phenomenon, the efficiency of

transfection of siRNA is quantitatively evaluated by the WST-8

method (a method for determination of viable cell count).

Effect of Eg5 siRNA（%）=（1 – Eg5 siRNA A450 / Non-targeting siRNA A450）×100

Jurkat U-937 THP-1 HeLa

3% 0% 3% 47%

30% 1% 45% 74%

73% 94% 94% 87%

0.0

0.4

0.8

1.2

1.6

2.0

Control(PBS) Product X Product R GenomONE-si

Raji Negative control siRNA

CDC2 siRNA

GenomONE - Si

■■■■ Cyclophilin B miRNA transfection in mouse primary T cell

■■■■ Comparison with competitors

One day after stimulation of mouse splenocytes with PMA/ionomycin, T cells were isolated. Two days after transfection of

cyclophilin B miRNA using GenomONE-Si, mRNA expression of cyclophilin B was measured by qRT-PCR. Furthermore, the knockdown

was also confirmed by the Western blotting.

■■■■ Successfully transfected cellsHuman primary monocyte, Human primary T cell, Normal human epidermal melanocyte, Mouse primary B cell,

Mouse primary T cell, Mouse primary macrophage, Mouse primary granulosa cell, Mouse primary alveolar type 2 cell,

Mouse primary mast cell, Rat primary cardiac myocyte, Rat primary granulosa cell, Ramos, RAW264.7, Jurkat, K-562,

U-937, THP-1, HL-60, Raji, HeLa, HeLa S3, J774A.1, Reh, YN-1, TF-1a, C2C12, HuH-6, HuH-7, Hep G2, Caco-2, HT-29,

SW-480, SW-620, HCT 116, SK-CO-1, COLO 201, A549, TE-13, Du-145, MIN6, NIH-3T3, INS-1E, MIA PaCa-2, ME-180,

H1299, MCF-10A, mpkCCD(c14), U251 MG, D54 MG, HMVEC-dLyNeo, HUVEC, CMK6G3(Monkey ES cell)4

Eg5 siRNA

Relative Quantity; qRT-PCR assays (n=3)

74%86%

High knockdown effects were obtained using GenomONE-Si, whereas no adequate effect could be obtained using other transfection

reagents; thus, the superiority of GenomONE-Si was demonstrated.

CDC2 siRNA

a; Negative control miRNA (1000pM)b; Cyclophilin B miRNA (1000pM)

c; Cyclophilin B miRNA (400pM)d; Cyclophilin B miRNA (100pM)

e; Cyclophilin B miRNA (25pM)

f ; Cyclophilin B miRNA (6.3pM)

β-actin

Cyclophilin B

a b c d e f

Western blotting

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10

20

30

40

50

60

70

80

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control(PBS)

400pM 400pM 100pM 25pM 6.3pM

N.C.miRNA

Cyclophilin B miRNA

Ce

ll Via

bility

(%)

Re

lative

Qu

an

tity

(no

rma

lize

d 1

8S

rR

NA

)

Relative Quantity Cell Viability(%)

75%

56%

Product X

Product R

GenomONE-Si

Re

lati

ve

Qu

an

tity

(no

rma

lize

d 1

8S

rR

NA

)

Re

lati

ve

Qu

an

tity

(no

rma

lize

d 1

8S

rR

NA

)

GenomONE-SiGenomONE-Si

®

・Optimal for transfection of synthetic oligo-type siRNA/miRNA

・Applicable to difficult-to-transfect immune cells

・Optimal for rapid screening of a large number of test samples (high-throughput screening)

KALA peptides can destabilize the endosomal membrane and

enhance the transfection efficiency of non-viral gene delivery

vectors.

KALA peptide: WEAKLAKALAKALAKHLAKALAKALKACEA

GenomONE - GX・A novel gene delivery vector composed of a lipid-like substance and HVJ-E

・The GenomONE-GX Enhancer suppresses innate immune responses by plasmid DNA and

improves transgene expression.

・Transgene expression is enhanced by GenomONE-GX using a combination of KALA peptides.

■■■■ TurboGFP expression

Various cell lines were transfected with a reporter plasmid encoding TurboGFP under a CAG promoter using either GenomONE-GX(with or without the KALA peptide) or Product L (a competitor), following the manufacturer’s instructions.

※The KALA peptide is not included in the GenomONE-GX kit and hence the KALA peptide solution needs to be prepared in advance.

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GenomONE-GX

Product L

(competitor)

Jurkat K-562 RAW 264.7 HEK-293 RIN-5F SH-SY5Y

GenomONE-GX

+KALA peptide

■■■■ Mechanism of Enhancer

The GenomONE-GX Enhancer inhibits innate immune signaling

in response to the recognition of exogenous DNA. Hence, it

can be used to increase transgene expression, which is

intrinsically suppressed by innate immune signaling.

■■■■ Successfully transfected cellsiPS, HAEC, MEF, MRC-5, WI-38, Jurkat, K-562, P3X63Ag8.653, RAW 264.7, THP-1, U-937, 3T3-L1, A549, CHO-K1,

HEK-293, HeLa, HeLa S3, Hep G2, Hs68, HuH-7, L6, L929, MCF-7, Neuro-2a, NIH-3T3, RIN-5F, SAS, SH-SY5Y

■■■■ Mechanism of KALA peptide

Gene delivery vector

Nucleus

Plasmid DNADNA sensor

Enhancer

Plasmid DNAGene delivery vector

Cytoplasm

KALA peptide

Endosomal escapeEndosome

IFN-β

Inflammatory cytokines

Cytoplasm

®

ST striated duct, G:granular convoluted tubule, A:acini

non-injection side rCLCA siRNA-injection side

GenomONE - Neo・Applicable to in vivo transfection (plasmid DNA, siRNA/miRNA, and protein)

・Optimal for transfection of protein in vitro

■■■■ Retrograde Injection of siRNA into Rat Submandibular Gland（in vivo）

rCLCA siRNA was injected in retrograde fashion into the

submandibular glands of rats, using GenomONE-Neo .

Forty-eight hours later, specific suppression of expression of

Cl- channel protein was demonstrated on the siRNA-

injection side by immunostaining.

rCLCA siRNA was injected in retrograde fashion into the

submandibular glands of rats, using GenomONE-Neo.

Forty-eight hours later, secreted saliva was collected while a

drug stimulating salivary release (pilocarpine) was administered.

Analysis of the concentrations of electrolyte in the saliva

collected revealed a significantly higher Cl- level in the group

with injection of rCLCA siRNA than in the control group

(scrambled siRNA-injection group), confirming the efficacy of

rCLCA siRNA injection in specifically suppressing Cl-

reabsorption. Reabsorption of Na+ and K+ remained unaffected.

Injection of siRNA, targeted at cystic fibrosis transmembrance conductance regulator (CFTR), also resulted in specific suppression of

Cl- reabsorption.

[Data] Dr. Kazunari Ishibashi

Department of Functional Bioscience, Fukuoka Dental College (Japan).

[Related article] K. Ishibashi et al., J. Dent. Res., 85 (12), 1101-1105 (2006).

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■■■■ Intracellular delivery of Cre recombinase (in vitro)

By introducing Cre recombinase (protein) into 2-2 cells with GenomONE-Neo, loxP sites inserted in the genome sequence were

deleted and LacZ gene expression was induced.

Suppression of Cl- reabsorption in submandibular gland

Specific suppression of expression of Ca2+-dependent Cl- channel protein (rCLCA)

Immunostaining

®

2-2 (monkey, African green, RIKEN BioResource Center):

35 copies of PCAG-loxP-neopA-loxP-LacZ were tandemly inserted into the genome.

Cre recombinase

alone

HVJ-E

+Cre recombinase

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GenomONE - CF・A cell fusion reagent with low cytotoxicity and

high fusion activity・Applicable to hybridoma preparation and

nuclear transfer・An alternative method to PEG and electrofusion for cell fusion

■■■■ Comparison with PEG method in the fusion of different types of cell

■■■■ Comparison with PEG method in hybridoma preparation

Rat MSC cells (rat bone marrow-derived mesenchymal stem cells) labeled with red fluorescence were combined with rat primary

cardiac myocytes labeled with green fluorescence in Cell Fusion Buffer. As a result, fused cells (yellow) were formed (GenomONE-CFsuspension method). Fused cells adhering to the plate were also observed after 1-2 days of culturing. In the PEG-treated group, high

cytotoxicity appeared immediately after cell fusion, reducing the number of fused cells obtained.

Normal BALB/c mouse splenocytes not sensitized with antigen were fused to X63-Ag8.653 myeloma cells using GenomONE -CF or

PEG1500. Beginning the following day, half of the culture medium (10%FBS/RPMI1640) was replaced with HAT medium at five

points of time(Days 1, 2, 3, 5, and 8), and the growth of colonies in each well was assessed on Days 10 - 11 to determine the

hybridoma-positive rate(an indicator of efficiency of fusion). On Day 12, mouse antibody level (IgG + IgA + IgM) in the supernatant

was measured by ELISA, to calculate the antibody production-positive rate. The effect of adding a commercially available hybridoma

supplement to the medium after fusion was also assessed (supplement was also added to the HAT medium).

Use of GenomONE -CF resulted in more efficient formation of antibody-producing hybridoma than that of PEG. The efficiency of cell

fusion mediated by GenomONE -CF was increased by the addition of hybridoma supplement to the medium used for incubation after

cell fusion.

Immediately after mixing

GenomONE-CF

PEG

®

After one day of culturing After 2 days of culturing

Hybridoma supplement

Hybridoma-positive rate

Antibody production-

positive rate

GenomONE -CF

－

－－

－38/96 (40%) 9/96 (9%)

＋

＋＋

＋96/96 (100%) 96/96 (100%)

PEG1500－

－－

－3/96 (3%) 1/96 (1%)

＋

＋＋

＋36/96 (38%) 9/96 (9%)

GenomONE series

Product selection guide

Product information

Visit our website for more detailed information regarding applications,

safety data sheets, or for any other inquiry.

https://www.iskweb.co.jp/eng/products/hvj-e/

2019.5

®

GenomONE-GE GenomONE-Si GenomONE-GX GenomONE-Neo GenomONE-CF

p. 3 p. 4 p. 5 p. 6 p. 7

Cas9 protein,

gRNA 〇 △ ― △ ―

plasmid DNA ― ― 〇 △ ―

siRNA/miRNA ― 〇 ― △ ―

Protein ― △ ― 〇 ―

in vivo transfection ― △(siRNA/miRNA)

― 〇 ―

Cell fusion ― ― ― ― 〇

in vitro

transfection

〇：Highly Recommended △：Applicable/Not Recommended －：Not Applicable

ISHIHARA SANGYO KAISHA, LTD.

1-3-15, Edobori, Nishi-ku, Osaka 550-0002 JAPAN

Manufacturer

URL: https://www.iskweb.co.jp/eng/products/hvj-e/

E-MAIL: HVJ-E@iskweb.co.jp

These products are sold for research purpose only. It may not be used for treatment or other clinical purposes or for intra- and extracorporeal diagnosis in humans or animals.