genomone-gx +kala peptide - iskweb.co.jp · 3-15 edobori 1-chome, nishi-ku, osaka 550-0002...

Effect of KALA peptide (TurboGFP expression)

※ Note: KALA peptide is not included in GenomONE-GX kit

Two days after transfection, TurboGFP expression was detected via fluorescent microscopic analysis. GenomONE-GX with the addition of KALA peptide exhibited a higher transfection efficiency than product L(competitor).

One day after transfection, luciferase expression was quantified.

KALA peptide upregulated luciferase expression in many cell types.

1.Gene transfer vector/pDNA/KALA peptide complex

is internalized via endocytosis.

2. KALA peptide induces membrane pore formation.

3. pDNA is released into cytoplasm.

Mechanism of KALA peptide-mediated endosomal escape

Overview of transfection using GenomONE-GX +KALA peptide Effect of KALA peptide (luciferase expression)

GenomONE-GX +KALA peptideInstruction manual

GenomONE-GX kit

KALA peptide : WEAKLAKALAKALAKHLAKALAKALKACEA

Ver.2 July 2019

GenomONE –GX

GenomONE –GX

+KALAペプチド

Product L(competitor)

Jurkat

JurkatJurkat

Jurkat K

KK

K-

--

-562

562562

562 RAW 264.7

RAW 264.7RAW 264.7

RAW 264.7 HEK

HEKHEK

HEK-

--

-293

293293

293 SH

SHSH

SH-

--

-SY5Y

SY5YSY5Y

SY5YRIN

RINRIN

RIN-

--

-5F

5F5F

5FA549

A549A549

A549 MCF

MCFMCF

MCF-

--

-7

77

73T3

3T33T3

3T3-

--

-L1

L1L1

L1 HeLa S3

HeLa S3HeLa S3

HeLa S3 L929

L929L929

L929 NIH/3T3

NIH/3T3NIH/3T3

NIH/3T3L6

L6L6

L6Hs68

Hs68Hs68

Hs68U

UU

U-

--

-937

937937

937

* KALA peptide is not included in the GenomONE-GX kit. Please prepare the KALA peptide solution in advance.

* Prepare 6 wells of cells so that both settling time on ice (Step ⑥) and the effect of Enhancer

(Step ⑦) can be determined.

* Prior to vector preparation, vortex Reagent 3 quickly for 2 or 3 seconds.(Do not spin down)

* Cool all the reagents on ice before use. Be sure to perform Steps ① through ⑥ on ice.

* The protocol described below is suitable only for evaluating transfection conditions.

Once transfection conditions are established, prepare the vector in sufficient amounts to maintain

a stable mixing ratio with each of the reagents used in Steps ① to ⑥.

Add Enhancer to only one of the two

wells treated with each gene transfer vector prepared with settling time of 1, 3, and 5 min.

(×3 wells)

Reagent 1 taken into a micro-test tube

Add Reagent 2 and mix well

(by pipetting).

Add 0.2mg/mL DNA TE solution and

mix well (by pipetting).

Add Reagent 3 and mix well

(by pipetting).

Add Reagent 4, mix well (by pipetting),

and allow to stand on ice. Add the mixture (gene transfer vector) into two wells after 1 min, two wells after 3 min, and two wells after 5min.

(1 min: ×2 wells, 3 min: ×2 wells, 5 min: ×2 wells, total 6 wells)

Step

⑦

Step

①

Step

②

Step

③

Step

④

Step

⑥

GenomONE-GX +KALA peptideProtocol for preparation of gene transfer vector

2.5μL

（×3 wells）

1μL

6μL

6μL

6μL

Reagent 4 : 6μL

Gene transfer vector :1 min 5μL(×2 wells)3 min 5μL(×2 wells)5 min 5μL(×2 wells)

Add 0.02mg/mL KALA peptide and

mix well (by pipetting).

Step

⑤

6μL

96-well plate

Determine the amount to be added according to the

table below.

※For Corning's cell culture vessels

For first use, prepare a solution of Reagent 1 in

ethanol.(Add 500μL of ethanol and mix well via

vortexing to dissolve completely)

To control for the effect of KALA peptide, use sterile

water instead of KALA peptide in Step ⑤.

If cytotoxicity is observed, increase cell density and/or

reduce the on-ice settling time in Step ⑥.

If sufficient cell number for 6 wells can not be prepared,

settling time on ice in step ⑥ should be 3 min.

Use of Reagent 5 instead of Reagent 3 may achieve

higher gene expression levels in some cell lines. For

reference, visit our website.

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

In Step ⑥, the settling time on ice can alter transfection efficiency. The optimal settling time varies, depending

on the cell type; hence, determine the optimal time,

which is generally between 1 and 5 min.

If no cytotoxicity occurs, the amount of the vector

prepared in Step ⑥ and to be added to cells may be

increased up to 3 times. For more specific amounts to

be added, see table above.

If the effect of the Enhancer is poor, increase the

amount of Enhancer within the range shown in above

table. If Enhancer‘s effect is not obtained, omit Step ⑦.

●

●

●

●

●

●

●

●

●

Step ⑥

settling time on ice

1 min 3 min 5 min

Ste

p ⑦

En

ha

nce

r

(-)

(+

)

×6 well

24-well plate 6-well plate

×2 wells

on ice

1 min 3 min 5 min

×2 wells×2 wells

×3wells

12.5μL

（×3 wells）

5μL

30μL

30μL

30μL

Reagent 4 : 30μL


30μL

50μL

（×3 wells）

20μL

120μL

120μL

120μL

Reagent 4 : 120μL


120μL

96-well 0.1 0.32 1 5-15 2.5-3

48-well 0.2 0.95 3 10-30 5-6

24-well 0.5 1.9 6 25-75 12.5-15

12-well 1 3.8 12 50-150 25-30

6-well 2 9.5 30 100-300 50-60

Step ⑦

Amount of

Enhancer

(μL)

Amount of gene transfer vector

and Enhancer to be added per well

Plate size

Typical total

volume of

medium

(mL)

Growth area※

(cm2)

Relative area Step ⑥

Amount of gene

transfer vector

(μL)

Influence of settling time on ice on luciferase gene expression

Recommended transfection conditions for various cultured cellsEffect of KALA peptide for various promoters

E-mail : [email protected]

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

ISHIHARA SANGYO KAISHA, LTD.

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

Different culture conditions (number of passages, composition of medium, serum lot etc.)

are predicted to result in different transfection efficiencies. In order to obtain optimal results, it is necessary to consider detailed transfection conditions by referring to above table.

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

3.0E+06

3.5E+06

KALApeptide

(-)

KALApeptide

(+)

KALApeptide

(-)

KALApeptide

(+)

KALApeptide

(-)

KALApeptide

(+)

KALApeptide

(-)

KALApeptide

(+)

HEK-293 Hep G2 RAW 264.7 K-562

Lucifera

se e

xpre

ssio

n (

RLU

)

1 min 3 min 5 min 7 minStep ⑥ settling time on ice

Cell

CellCell

Cell

Number

NumberNumber

Number of cell

of cellof cell

of cell

(96

(96(96

(96-

--

-well plate)

well plate)well plate)

well plate)

Step

StepStep

Step④

④④

④ Step

StepStep

Step⑤

⑤⑤

⑤

KALA peptide

KALA peptideKALA peptide

KALA peptide

Step

StepStep

Step⑥

⑥⑥

⑥

settling time

settling timesettling time

settling time

on ice

on iceon ice

on ice

Step

StepStep

Step⑥

⑥⑥

⑥

Amount

AmountAmount

Amount of

of of

of

gene transfer

gene transfer gene transfer

gene transfer

vector to be

vector to be vector to be

vector to be

added per well

added per welladded per well

added per well

Step

StepStep

Step⑦

⑦⑦

⑦

Enhancer

EnhancerEnhancer

Enhancer

A549 5×10

3

cells Reagent 3 + 3分 5μL -

CHO-K1 5×10

3


HEK-293 1.5×10

4

cells

Reagent 3

Reagent 3

-

+

5分

1分

5μL

5μL

-

-

HeLa

5×10

3

cells

1×10

4

cells

Reagent 5

Reagent 5

-

+

3分

1分

5μL

5μL

-

-

HeLa S3 1×10

4

cells

Reagent 3

Reagent 3

-

+

3分

1分

5μL

5μL

+

+

Hep G2 1.5×10

4

cells Reagent 5 + 5分

5μL

+

Hs68 1×10

4

cells Reagent 5 + 3分 5μL +

HuH-7 5×10

3


Jurkat 4×10

4


K-562 2×10

4


L6 5×10

3


L929 5×10

3


MCF-7 2×10

4


NIH/3T3 5×10

3


P3X63Ag8.653 4×10

4


RAW 264.7 2×10

4

cells

Reagent 5

Reagent 5

+

+

3分

1分

5μL

10μL

+

+

RIN-5F 4×10

4


SH-SY5Y 5×10

4


THP-1 4×10

4

cells Reagent 3 - 5分 5μL +

U-937 2×10

4

cells

Reagent 3

Reagent 5

+

+

3分

1分

5μL

15μL

+

+

3T3-L1 5×10

3


For adherent cells, cells should be plated one day before transfection.

genomone-gx +kala peptide - iskweb.co.jp · 3-15 edobori 1-chome, nishi-ku, osaka 550-0002...

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