novel transfection system nepa21 · ex vivo (retina) 34 cross section montage of 2-photon (2p)...

NOVEL TRANSFECTION SYSTEM NEPA21

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ABOUT NEPA GENE CO., LTD.

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About Nepa Gene Co., Ltd.

• Founded：February, 2001

• CEO：Yasuhiko Hayakawa

・・・With over 20 years of experience in the field of electroporation

and cell fusion, he founded Nepa Gene Company.

• The current main business is manufacturing & distributing all over the world

– Gene Transfection Systems

– Electro Cell Fusion Systems

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ELECTROPORATOR

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What is Electroporator?

• Generate Electroporation (EP) by Applying Electric Pulses

– The Formation of Temporal Pores (Small Holes)

– The Transfer of Materials into Cells

Before EP During EP After EP

Pores form (small holes)

Cell Membrane

DNA/RNA transfer

Pores reseal

Cell Membrane Cell Membrane 5

Electrodes

Square Wave Pulse

Direct Current

• Mammalian Cells • In Vivo

Conventional Electric Pulses

Exponential Decay Pulse

Direct Current

• Bacterial Cells • Fungal Cells • Plant Cells (Not good for Mammalian Cells)

V V

Time (ms or μs) Time (ms or μs)

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+ Special Buffers

•High poring efficiency •Low viability

• Mild poring • High viability • May not work well with Bac- terial Cells

NEW ELECTROPORATOR NEPA21

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NEPA21 Novel 3-Step Multiple Pulses

Time

0V

Vo

ltag

e

Step 3: Polarity Reversed Transfer Pulses

For increasing the transfection efficiency.

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• High Transfection Efficiency

• High Viability

• Without Special Buffers

+ -

Step 1 • High Voltage • Short Duration • Voltage Decay

Step 2 • Low Voltage • Long Duration • Voltage Decay

Step 3

Polarity Reversed • Low Voltage • Long Duration • Voltage Decay

Step 1: Poring Pulses

For forming pores (small holes) in cell membrane with low damage.

+ -

Step 2: Transfer Pulses For delivering the target molecules into cells with low damage.

+ -

NEPA21 Features

• High Transfection Efficiency & High Viability Without Special Buffers

• A Wide Range of Applications

– Cells Not Only In Suspension But Also In Adherent Status

– Tissue Slices / Organs (Ex Vivo)

– In Vivo, In Utero and In Ovo

• Displaying Useful Electric Parameters

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Measureing Ω between Electrodes

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• Target Cells/Tissues • Buffers • Electrode Material/Quality/etc. • Etc.

Voltage = Current (A) x Resistance (Ω)

Depending on

Fluctuating

NEPA21 can measure impedance* (Ω) between electrodes. Setting voltage can be optimized by the Ω check before pulsing.

*The measured impedance is almost the same as the resistance.

Controllable by machine settings

Displaying of Pulse Parameters

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Electroporators Objects Parameters

NEPA21 Electroporator All Pulses Voltage, Ampere, Time & Joule*

CUY21 Electroporators (Nepa Gene’s Previous Models)

One Pulse Only Voltage, Ampere & Time

The Other Company’s Electroporators

One Pulse Only Voltage & Time Only

Setting: 100V The Results will not be the same

*Joule (energy) = V x A x T

e.g

Sample A

Sample B

→ Impedance: 200Ω → Output Ampere: 0.5A

Setting: 100V → Impedance: 1000Ω → Output Ampere: 0.1A

User-Friendly Interface & Compact Unit

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NEPA21 CUY21SC (old-type Nepa device)

CUY21EDIT (old-type Nepa device)

NEPA21 APPLICATIONS

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A Wide Range of Applications

In Vitro

Bacterial Cells Fungal Cells

Animal Cells Plant Cells Other Cells

In Suspension

[Difficult-to-transfect Cells] Primary Cells Neurons Stem Cells Blood Cells Immune Cells etc.

Other Cell Lines

In Adherence

Tissues/Organs (Ex Vivo) Brain Slices, Embryos, etc.

In Vivo Mice, Rats, Chickens, etc. Brain, Eye, Muscle, Skin, Kidney, Liver, Testis, Embryo, etc.

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NEPA21 application range

Transfection into Cell Suspension

In Vitro



In Suspension


Other Cell Lines

In Adherence



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Cuvette Electroporation

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cross section

3-Step Multiple Pulses

Cells in Suspension

Transfection into Difficult-to-transfect Cells

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In Vitro



In Suspension


Other Cell Lines

In Adherence



Primary Cells

Primary Mouse Cerebral Cortex Neurons

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NEPA21 With Electroporation Cuvettes

Viability: 80% Transfection Efficiency: 70%

9 days after electroporation Red: MAP2 Blue: GFAP Green: GFP

The cells were from E14 Mouse Cerebral Cortex

Primary Cells

Primary Mouse Cerebellar Granule Neurons

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Viability: 91% Transfection Efficiency: 65%

Primary Cells

BMMC Mouse Bone Marrow-Derived Mast cells HASM Human Airway Smooth Muscle cells

Mouse Embryonic Fibroblasts (MEF) Human T Cells

V: 80% TE: 83%

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V: 90% TE: 80%

V: Viability, TE: Transfection Efficiency

V: 90% TE: 85% V: 58% TE: 90%

V: 20% TE: 60% Nucleofector

Primary Cells

Primary Mouse Embryonic Fibroblasts (MEF)

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Neon (Invitrogen) NEPA21 (Nepa Gene)

Shanghai Jiao Tong Unversity

Stem Cells, Immune Cells, Etc.

Mouse ES cells Primary Human Mesenchymal Stem cells

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NEPA21 With Electroporation Cuvettes V: Viability, TE: Transfection Efficiency

V: 78% TE: 75%

Jurkat Human T-cell Leukemia cells BJAB EBV-negative Burkitt Lymphoma cells

V: 90% TE: 85% V: 96% TE: 96%

Nucleofector

V: 20% TE: 20%

Human iPS cells

Mouse ES Cells

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Transfection into Other Cell Lines in Suspension

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In Vitro



In Suspension


Other Cell Lines

In Adherence



Cell Lines (1)

Cell Line V TE

Species: Human

HeLa Human Cervical Carcinoma cells 95% 95%

293 Human Embryonic Kidney cells 90% 90%

293T Human Embryonic Kidney cells 90% 90%

TIG-7 Human Embryonic Lung Fibroblasts 89% 76%

MRC-5 Human Embryonic Lung Fibroblasts 85% 90%

SUSM-1 Human Fibroblasts 77% 71%

KMST-6 Human Fibroblasts 70% 60%

HT1080 Human Fibrosarcoma cells 93% 81%

MIA-PaCa-2 Human Pancreatic Carcinoma cells 80% 77%

HepG2 Human Hepatoma cells 80% 76%

HuH-7 Human Hepatoma cells 70% 60%

H1299 Human Lung Cancer cells 90% 90%

HSC-2 Human Squamous Carcinoma cells 93% 98%

HSC-3 Human Squamous Carcinoma cells 93% 98%

OVCAR-3 Human Ovarian Carcinoma cells 90% 79%

RMG-1 Human Ovarian Carcinoma cells 97% 67%

Cell Line V TE

Species: Human

TE-1 Human Esophageal Carcinoma cells 85% 41%

TE-8 Human Esophageal Carcinoma cells 85% 40%

MCF-7 Human Breast Cancer cells 90% 93%

T47D Human Breast Cancer cells 90% 85%

NUGC-3 Human Gastric Carcinoma cells 73% 68%

A549 Human Lung Adenocarcinoma cells 85% 90%

LNCaP Human Prostate Carcinoma cells 71% 90%

RKO Human Colorectal Carcinoma cells 80% 40%

SK-N-SH Human Neuroblastoma cells 95% 95%

SH-SY5Y Human Neuroblastoma cells 60% 90%

KG-1-C Human Oligodendroglial cells 85% 60%

HaCaT Human Keratinocyte cells 40% 80%

CCD1079sk Human Skin Fibroblasts 40% 53%

iHAM-4 Human Amniotic Mesenchymal cells 59% 95%

iHAM-7 Human Amniotic Epithelial cells 70% 40%

HTR-8/Svneo Human Trophoblast cells 95% 67% Jurkat Human T-cell Leukemia cells 90% 85% Namalwa Human Burkitt's Lymphoma cells 70% 75%


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NEPA21 with Electroporation Cuvettes

Cell Lines (2)

Cell Line V TE

Species: Human

Raji Human Burkitt's Lymphoma cells 97% 83%

LCL Human Lymphoblastoid cell 55% 43%

Nalm-6 Human B-cell Precursor Leukemia cells 65% 63%

KU812 Human basophilic leukaemia cells 96% 42%

K562 Human erythroleukemia cells 91% 83%

MutuⅠ Human Burkitt Lymphoma cells 87% 91%

MutuⅢ Human Burkitt Lymphoma cells 54% 92%

Species: Mouse

MEF Mouse Embryonic Fibroblasts 80% 90%

L Mouse L cells 90% 65%

MC3T3-E1 Mouse Osteoblastic cells 85% 75%

MS-1 Mouse Pancreatic Endothelial cells 90% 90%

RAW264.7 Mouse Macrophage-like cells 70% 56%

WR19L Mouse T-cell lymphoma cells 92% 60%

3T3-L1 Mouse Preadipocytes 90% 90%

XS106 Mouse Dendritic cells 61% 45%

Cell Line V TE Species: Mouse mDC Mouse Myeloid Dendritic cells 79% 72% MC/9 Mouse Mast cells 76% 84% TS Mouse Trophoblast Stem cells 59% 47% Species: Rat

PC12 Rat Adrenal Pheochromocytoma cells 70% 50%

H9c2 Rat Ventricular Myoblasts 71% 82% REF Rat Embryonic Fibroblasts 90% 99% C6 Rat Glioma cells 80% 67% Species: Hamster CHO Chinese Hamster Ovary cells 74% 90% CHO-K1 Chinese Hamster Ovary cells 95% 95% Species: Dog MDCK Chicken B cells 90% 95% Species: Chicken DT40 Chicken B cells 91% 38%


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NEPA21 with Electroporation Cuvettes

Disposable Kits / Special Buffers NEPA21 (Nepa Gene) Nucleofector (Lonza) Neon (Invitrogen)

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Cuvettes Special Buffers

Tips Special Buffers

Cuvettes Only No Special Buffers

The Cost of One Electroporation:

JPY 210


JPY 2,750


JPY 1,719

Transfection into Cells in Adherence

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In Vitro



In Suspension


Other Cell Lines

In Adherence



Electrode

5mm Gap

Bottom of culture plate

7mm

5mm Gap 5mm

Gap

CUY900-13-3-5

7mm

14mm

Special Electrode for Adherent Cells

Cells in adherence

(Electric pulses are delivered)

Primary Neurons In Adherence

NEPA21 With Adherent Cell Electrode

CUY900-13-3-5 Adherent Cell Electrode

EGFP fluorescence image of the neurons 2 days after electroporation

Transfection into primary neurons cultured for 6 days in adherent state.

The neurons were prepared from E15 mouse cerebral cortex.

High magnification image of Figure B.

Many robust EGFP signals suggest high transfection efficiency.

High magnification image of Figure C (x40)

Neurites are shown clearly.

Department of Neurochemistry, National Institute of Neuroscience, Japan 30

A

Primary Mouse Microglial cells In Adherence

Electroporation: After 1 day in vitro (DIV) on 24-well plates, post 1-week co-culturing astrocyte and microglial cells

Transfection into Tissues/Organs

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In Vitro



In Suspension


Other Cell Lines

In Adherence



Ex Vivo (Tissue Fragments)

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Electroporation-mediated expression of fluorescent proteins in hippocampal neurons. (a-c) Organ culture of hippocampal tissue fragments three days after electroporation with CAG-eGFP (a),Ta1X4 –eGFP (b), and b-actin-eGFP (c) expression constructs. (d,e) A mature hippocampal neuron maintained 14 days in dissociated culture after electroporation of a b-actin-eGFP expression construct. Higher magnification view of the region marked by a rectangle in (d) reveals dendritic spines on the surface of dendritic shafts (arrows in e).

cross section

CUY701P2E / CUY701P2L

Kawabata I et al., Electroporation-mediated gene transfer system applied to cultured CNS neurons., Neuroreport. 2004 Apr 29;15(6):971-5.

Ex Vivo (Retina)

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cross section

Montage of 2-photon (2P) micrographs of whole-mounted mouse retina Green: Oregon Green 488 BAPTA-1 [OGB-1; focal plane in ganglion cell layer (GCL) , only green channel shown] Red: sulforhodamine 101 (SR101)


Briggman KL, et al., Bulk electroporation and population calcium imaging in the adult mammalian retina., J Neurophysiol. 2011 May;105(5):2601-9.

Ex Vivo (Early Chick Embryos)

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(a) A stage 5 chick embryo electroporated with pCAGGS-GFP plasmid in vitro. (b–d) The embryo under an epi-fluorescent dissecting microscope at stage 6, 10, and 17. The neural tube and some head ectoderm express GFP.


Hatakeyama J et al., Method for electroporation for the early chick embryo. Dev Growth Differ. 2008 Aug;50(6):449-52.

Ex Vivo (Mouse Embryonic Kidney and Lung)

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Kidney Lung

Bath w/platinum plate electrodes on petridish, 5mm gap

CUY520P5

Courtesy of Matsumoto S, Graduate School of Medicine, Osaka University, Japan

In Vivo Transfection

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In Vitro



In Suspension


Other Cell Lines

In Adherence



In Utero (Mouse Embryos)

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One uterine horn is drawn out through a hole in a piece of sterile gauze.

Plasmid DNA was injected into one or both lateral ventricles.

The presence of Fast Green made the shape of the lateral ventricles visible (arrows).

electric pulses were applied to the brain from outside the uterine wall

A

GFP expression after EP. Embryonic day 14.5 (E14.5) mouse embryos were subjected to in utero EP and fixed 2 days later. The brains were taken out and observed under a fluorescence stereomicroscope (A).

Changes in the position and morphology of GFP-positive cells after in utero EP (B–F). E16 (B) or E15 (C–F) mouse embryos were transfected with CAG-EGFP, and fixed 12 h (B), 1.5 days (C), 2.5 days (D), 3 days (E) and 6 days (F) after EP. CP; cortical plate, IZ; intermediate zone, MZ; marginal zone; SVZ; subventricular zone; VZ; ventricular zone. Bar, 50 μm.

CUY650P5

Tabata H et al., Labeling embryonic mouse central nervous system cells by in utero electroporation. Dev Growth Differ. 2008 Aug;50(6):507-11.

Barnabé-Heider F et al., Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation., Nature Methods. 2008 Feb;5(2):189-96.

In Vivo (Brain)

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CUY200S

CUY650P5

Periaqueductal gray (PAG)

Inoue M et al., Locus-specific rescue of GluRepsilon1 NMDA receptors in mutant mice identifies the brain regions important for morphine tolerance and dependence., J Neurosci. 2003 Jul 23;23(16):6529-36.

Dentate gyrus of the hippocampus

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In Vivo (Retina)

Arrangement of electrodes for in vivo electroporation for RPE transfection. (A) Tweezer-type electrodes were placed on the corneal surface of either eye of a 1-month-old Sprague-Dawley rat. (B) The current was applied with the positive electrode contralateral to the injected eye. After prior injection of plasmid DNA into the subretinal space of the right eye, this arrangement electrophoresed the negatively-charged DNA toward the RPE layer (arrowheads).

The left eye and the optic nerve were exposed. A small hole was made with a 31-gauge needle 0.5 to 1.0 mm posterior to the limbus. The tip of the needle was viewed as it entered the vitreous at a 40° to 50° angle through the dilated pupil. 10 μg plasmid DNA in 4 μL TE buffer was injected into the vitreous. The eye was then immediately grasped between tweezerz-type electrodes, with the cathode attached to the corneal electrode and the anode with the slit attached to the scleral electrode.

CUY675P5 CUY650P7

In Vivo (Muscle)

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Histochemical staining for β-galactosidase activity in the muscle after gene transfer of pCAGGS-lacZ DNA with EP. The tibialis anterior muscle was injected with 50 µg of pCAGGS-lacZ plasmid DNA and treated with electric pulses of 100 V. Five days later, the muscle was excised. Transverse sections of the muscle sample were stained for β-galactosidase activity, and counterstained with eosin. Original magnification, × 40

Time course of IL-5 expression after transfer of pCAGGS-IL-5 plasmid by EP in vivo. The bilateral tibialis anterior muscles were injected with pCAGGS-IL-5 plasmid DNA. Electric pulses of 100 V were delivered to the DNA injection sites. Serum samples were obtained on the indicated days after EP and measured for IL-5 by ELISA. Each value represents the mean IL-5 concentration from three mice

CUY560-5-0.5

CUY650P3

Miyazaki S et al., In vivo DNA electrotransfer into muscle., Dev Growth Differ. 2008 Aug;50(6):479-83.

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siRNA (Subcutaneous Tumors)

To determine the optimal voltage for delivery of siRNAs into tumors, Cy3-labeled siSTABLE VEGF siRNA-SCR (red in Fig. 2A) was injected into the PC-3 tumors, and then the tumors were pulsed. After 24 h, the tumors were excised and frozen sections were observed under a confocal microscope system. Figure A shows that voltages ranging from 50 to 70 V were highly successful for delivering the siRNAs into the PC-3 tumors. Among the conditions, 70 V (91 V/cm) was the best to deliver the siRNAs into tumors.

CUY663P3X6

Takei Y et al., In vivo silencing of a molecular target by short interfering RNA electroporation: tumor vascularization correlates to delivery efficiency., Mol Cancer Ther. 2008 Jan;7(1):211-21.

In Ovo (Chick Embryos)

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CUY610P4-1

CUY613P1

In Vivo (Xenopus Embryos)

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Inject 5-10nl of GFP-mRNA（1μg/μl, 0.05% Fast Green）into intercellular space of upper-few epithelial layers of the neural plate, and immediately after injection (leaving the needle in the embryo) the electric shock（20-22V, 5msecON, 95msecOFF, 10 shocks） is given by the micro-electrode. A: Recognition of the target site by Fast Green just after EP. This dye fades away 10-15min later. B: Expression of GFP at the eye 15-20 hours later. C: Detection of GFP protein in the eye vesicle by FITC-conjugated immuno-staining of thin section. The local deliveries of mRNAs such as BMP and Shh result in the perturbation of eye development in addition to the up/down regulation of the eye-marker genes.

CUY195P0.5 CUY700P20E

Sasagawa S et al., Improved mRNA electroporation method for Xenopus neurula embryos., Genesis. 2002 Jun;33(2):81-5.

In Vivo (Zebrafish Embryos)

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Phalloidin staining of actin filaments with 3 dpf embryos. (A) The 3 dpf embryo injected with control MO has strong staining of phalloidin in the posterior macula (pm). (B) The embryo which was injected with control MO and then electroporated had similar levels of phalloidin staining in pm in the injection only embryos. (C) Injection with mif MOs into the otic vesicle alone did not cause reduction of phalloidin staining, while injection of mif MOs along with electroporation caused a dramatic reduction of phalloidin staining in the pm (D). am, anterior macula.

Holmes KE et al., Direct delivery of MIF morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development., J Vis Exp. 2011 Jan 7;(47).

Posterior macula (pm)

PUBLICATIONS

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THANK YOU

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novel transfection system nepa21 · ex vivo (retina) 34 cross section montage of 2-photon (2p)...

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