Transfection Fundamentals: Secrets to Success€¦ · Transfection Fundamentals: Secrets to Success Anjana Bhattacharya, PhD Mirus Bio LLC

Transfection Fundamentals:Secrets to Success

Anjana Bhattacharya, PhDMirus Bio LLC

2© 2012 Mirus Bio LLC. All rights reserved.

The Transfection Experts

TRANSFECTION

CHEMICAL TRANSFECTION

In Vivo DELIVERY

NUCLEIC ACID TRACKING

ELECTROPORATION


Pioneering Transfection1996-2012

• THE FIRSTS

• Low toxicity technology that led to the first serum compatible transfection reagent TransIT®-LT1

• First siRNA transfection reagent -TransIT-TKO®

• First serum compatible reagent for large RNA delivery - TransIT®-mRNA

Allternatives

• COMPLEMENTING TRANSFECTION

• Broad spectrum electroporation solution - Ingenio®

• Direct tracking of nucleic acid delivery - Label IT®

Emerging Solutions

2008-2012

• ALWAYS INNOVATING

• High performance animal free reagent - TransIT®-2020

• High yield protein production in suspension cells -TransIT-PRO®

• Biologically relevant transfection – 3D Transfection System

The Transfection Experts

BREAKTHROUGHS in TRANSFECTION


Transfection Fundamentals

Basic Mechanism of Transfection

Evaluating Transfection Performance

Striking the Expression-Toxicity Balance

Optimizing Transfection Output

Making Transfection Physiologically RelevantPrimary and stem cells; 3D transfection


What is Transfection?

(Not Drawn to Scale)


Transfection Methodologies

Reagent - based

CALCIUM PHOSPHATE

LIPIDS

•Cationic Lipids

•Liposomes

CATIONIC POLYMERS

•Polyethyleneimine

•Basic proteins/peptides

•Dendrimers

•DEAE-dextran

Instrument - based

ELECTROPORATION

OTHER

• Microinjection

• Biolistic delivery

• Magnetic transfectionVirus - mediated


Simple Transfection Reagent Protocol


What do we know about how Transfection occurs?







Why do we need Transfection Reagents?

(-) charge (-) charge

=

+

(+) charge

Neutralization of the negative charge& DNA condensation

(+) charge

Transfection Complex Formation



What happens during Transfection?


J Cell Biol. 2008 Jan 14;180(1):7-11

Transfection Complex


Cationic Lipid

Plasmid DNA

LIPOPLEXES

Net (+) Charge


O

O

O

NHO

Cl

N

+

Br

DOTAP - 1,2-dioleoyl-3-(trimethyammonium) propane

DDAB - Dimethyldioctadecylammonium(Bromide Salt)

What are Transfection Reagents made up of?Cationic Lipids


O

O

O

OHO

P

O

O

ONH3

HO

Me

Me

H

H H

Cationic Lipid + Helper Lipid

Plasmid DNA

LIPOSOME MULTI-LAYERED LIPOSOME


Net (+) Charge

DOPE - Dioleoylphosphatidylethanolamine

Cholesterol

What are Transfection Reagents made up of?Helper Lipids


What are Transfection Reagents made up of?Cationic Polymers

N+

NH

NN

N

NH2

NH2

NH2

(( ) ) )(( )

Quaternary amine

Primary amineSecondary amine

Tertiary amine

Branched PEI Linear PEI

Cationic Polymer

Plasmid DNA

POLYPLEX(DNA, Polycation)

Net (+) Charge


Very effective nucleic acid condensing agents


What are Transfection Reagents made up of?Lipid & Polymer Combinations

Lipid

Cationic Polymer

Plasmid DNA

LIPOPOLYPLEX(DNA, Lipid, Polycation)

Net (+) Charge


Synergistic combination – Effective condensation and complexation


Other features of Transfection Complexes

(+) charge

SIZE OF TRANSFECTION COMPLEXES

40 - 1000 nm

CHARGE

Transfection complexes should have a net cationic charge

Positive surface charge density - Zeta potential (+mV)

CHARGE RATIO

N+

NH

NN

N

NH2

NH2

NH2

(( ) ) )(( )

Quaternary amine

Primary amineSecondary amine

Tertiary amine

Branched PEI


Why Reagent: Nucleic Acid Ratio is important?

N/P ratio = Reagent concentration in Nitrogen residues (mM)

DNA concentration in Phosphate moieties (mM)

Reagent A Reagent B Reagent C


How to measure Transfection?








Reporter Systems

Green Fluorescein Protein (GFP)

Luciferase

- galactosidase

Secreted Alkaline Phosphatase(SEAP)


Visualising Nucleic Acid Delivery Directly

Labeling Nucleic Acid

• Fluorescent labeling

• Non fluorescent tags –Biotin, DNP


Retaining Function during Nucleic Acid Tracking

Very high labeling efficiencies can • Cause fluorophore quenching• Affect functionality

• Gene expression• Gene knockdown

MOLECULAR THERAPY Vol. 8, No. 2, August 2003

COS-7 cells Rhodamine-labeled DNA expressing nuclear YFP

HeLa cells Fluorescein labeled siRNA


How to get the best Experimental Output?








HeLa cells transfected with plasmid DNA encoding GFP


Sensitive Assays for Toxicity during Transfection

HeLa cells transfected with plasmid DNA encoding luciferase

Lactate Dehydrogenase (LDH) Assay )Measures membrane leakiness


What happens inside the cell?

HeLa cells transfected with non-coding plasmidGene expression profile assessed using

qRT-PCR based Human Stress Response 96 StellARray™


What happens inside the cell?


Which pathways are affected?


Different cell types show varied expression/toxicity profiles


Optimization is key to better results







Transfection WorkflowHow can we improve transfection performance?

Media

Nucleic Acid

Complex Formation Time

Cell confluence/density

Harvesting Time


Transfection Complex – Media Compatibility

Serum

Antibiotics

Polyanions

Pluronic® F68

Media

Media

Nucleic Acid



Harvesting Time

Transfection Inhibitors


Serum-free Media affects Transfection

0%

5%

10%

15%

20%

25%

30%

GFP

(%

)

NIH 3T3 cells transfected with a plasmid encoding GFP using TransIT®-LT1

Media


Protein yields are affected by Media

Applications where final cell densities is important:• Therapeutic Protein

Production - Antibodies Growth factors

CHO-S cells

Media


Factors affecting Transfection Performance

Media

Nucleic Acid



Harvesting Time

Nucleic Acid


The Quality of Nucleic Acid

Contaminants, such as protein, carbohydrate and lipids may affect transfection efficiency. Ideal plasmid preparation exhibits an A260/A280 ratio of > 1.8.

Endotoxin contaminated DNA inhibits transfection

COS-7 cells

Nucleic Acid

Use desalted siRNA. Use RNase free kits for purifying mRNA preps to preserve integrity.

http://en.wikipedia.org/wiki/File:LPS_en.svg


Nucleic Acid Dosage Matters

0.E+00

1.E+07

2.E+07

3.E+07

4.E+07

5.E+07

6.E+07

7.E+07

8.E+07

COS-7 CHO-K1 HEK 293

Lu

cif

era

se

Ac

tiv

ity

(R

LU

s)

3 µl reagent/1 µg DNA

1.2 µl reagent/0.4 µg DNA

TransIT®-LT1 Reagent

Nucleic Acid

COS-7 cells

TransIT-TKO® Reagent


Different Co-transfection Scenarios

Same Nucleic Acid

Multiple Plasmid DNAsMultiple siRNAsMultiple OligosMultiple mRNAs…

Always premix multiple nucleic acids to prevent preferential complexation with transfection reagent

Choose the molar ratio of nucleic acids carefully

Nucleic Acid


Different Co-transfection Scenarios

Different Nucleic Acids

Plasmid DNA + siRNA Plasmid DNA + Oligo Plasmid DNA + mRNA…

Mix complexes before adding to cells

Form complexes separately

Plasmid DNA siRNA

Nucleic Acid


Overexpressing Toxic Proteins

Lower plasmid dosage for the plasmid expressing toxic protein Add empty non-competing cloning vector, e.g. pUC18/19

HeLa cells co-transfected with a plasmidencoding luciferase and empty vector pUC18

Nucleic Acid



Media

Nucleic Acid



Harvesting Time

Complexing Time


Transfection Complex Formation Time

+ =

(-) charge (+) charge

0.E+00

4.E+07

8.E+07

1.E+08

2.E+08

2.E+08

0 10 20 30 40 50 60

Complex Formation Time (minutes)

Lu

cif

era

se A

cti

vit

y (

RL

Us)

CHO-K1 Cells transfected with a luciferase encoding plasmid using TransIT®-LT1 Reagent

0.E+107

4.0E+107

8.0E+107

1.2E+108

1.6E+108

2.0E+108

Complexing Time

0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

0 5 10 15 20

Luci

fera

se E

xpre

ssio

n (R

LUs)

Complex Formation Time (minutes)

CHO-K1 Cells transfected with a capped and polyadenylated luciferase transcriptusing TransIT®-mRNA Transfection Kit



Media

Nucleic Acid



Harvesting Time

Cell culture


Adherent cell confluency affects transfection

0.E+00

2.E+07

4.E+07

6.E+07

8.E+07

2 3 4 2 3 4

Lu

cif

era

se A

cti

vit

y (

RL

Us)

75% Confluency

100% Confluency

Amount of TransIT®-LT1 Reagent/µg DNA

NIH 3T3 cells transfected with a luciferase encoding plasmid using TransIT®-LT1

COS-7 cells

Cell cultureCell culture

Cell cultureCell culture

Cell culture


Suspension cell density impacts Transfection

CHO-S suspension adapted cells transfected with a GFP encoding plasmid using TransIT-PRO® Transfection Kit

Cell culture



Media

Nucleic Acid



Harvesting Time

Harvesting Time


Experimental goals determine harvest time

TYPICAL HARVESTING TIMES

Gene Over-expression 24 - 72 hours

Gene Knockdown 48 - 72 hours

mRNA translation 4 - 24 hours

Protein Production 2 - 14 days

Harvesting Time


Harvest times affected by nucleic acid turnover

Capped and polyadenylated firefly luciferase mRNA Delivered to NIH3T3 Cells using TransIT®-mRNA Transfection Kit

0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

3,500,000

4,000,000

0 6 12 18 24 30 36 42 48

Rel

ativ

e Li

ght

Un

its

Hours Post Transfection

Harvesting Time


Transfection Results Vary Day to Day

Compare transfection reagents on the same day Follow reagent specific protocols

Harvesting Time


Transfecting primary and stem cells







Primary and stem cells are relevant cellular models

Source : ATCC website

Transfection of mouse embryonic stem cells (mESCs) using TransIT®-LT1


Nucleic Acid Delivery - A tool for stem cell research

Stem cell reprogramming and differentiation


mRNA transfection of primary cells to generate iPS cells

BJ fibroblasts transfected with a modified base mRNA encoding GFP using TransIT®-mRNA Transfection Kit


Transfection of Human iPS cells

Human Induced Pluripotent (iPS) cells transfected with a ZsGreen encoding plasmid


Electroporation Schema

-+ -

+



When to choose Electroporation?

ADVANTAGES– Most efficient method for hard to

transfect cells and non-dividing primary cells

– Universal method for delivering all types of nucleic acids

– Effective on a broad range of cell types

DISADVANTAGES– Requires

Electroporation equipment and cuvettes

Larger numbers of cells Larger amounts of nucleic acid

– Higher cell death– Not suitable for short term (<6 hours)

expression experiments


Electroporation Workflow and Optimization

Harvest cells

Wash cells and resuspend in Ingenio Solution

Cells

Nucleic Acid

Electroporation Solution

Pulse Conditions

Mix DNA with cells, transfer to cuvette

Electroporate with optimal pulse

Transfer electroporated cells toculture vessel, incubate and harvest


Electroporation Solution determines Expression/Toxicity


Ingenio® Electroporation Solution is comparable to Amaxa®


Transfection of iPS derived iCell® Cardiomyocytes

High efficiency GFP plasmid transfection of iCell® Cardiomyocytesusing TransIT®-LT1 Transfection Reagent

Efficient knockdown of GAPDH expression in iCell® Cardiomyocytesusing TransIT-TKO® Transfection Reagent


More biologically relevant systems - 3D Transfection

alvetex® 12-well plate alvetex® scaffold cross-section 3D growth of HaCaT cells in alvetex


More biologically relevant systems - 3D Transfection

GFP expression in a cross-section of NIH 3T3 cells grown and transfected in an alvetex® 12-well plate

Luciferase expression in a variety of cell types grown and transfected in alvetex® 12-well plates


Experimental Goal – Single most decisive factor

Set-up• Transfection -or- Electroporation

• Different Nucleic Acids

• Gene Over-expression

• Gene knockdown

• Co-transfections???

Output• Imaging

• Flow cytometry

• Western Blots

• Reporter Assays

• QPCR


Find the videos at our YouTube page


www.TheTransfectionExperts.com

http://www.thetransfectionexperts.com/


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Transfection Fundamentals: Secrets to Success€¦ · Transfection Fundamentals: Secrets to Success Anjana Bhattacharya, PhD Mirus Bio LLC