transfection guidelines
TRANSCRIPT
© 2007 Mirus Bio Corporation. All rights reserved.
Transfection and RNAi
Technologies
B. Gopalakrishnan, Ph.D.
Mirus Bio Corporation
Madison, Wisconsin
USA
© 2007 Mirus Bio Corporation.
All rights reserved.
2
Mirus Bio Corporation
• Madison, WI- USA based biotechnology company
founded by Jon Wolff and V. Budker in 1995 that
specializes in non-viral nucleic acid delivery
• Therapeutic pipeline
– Develop efficient, non-viral gene delivery technologies
• Focused on understanding the basic mechanisms of cellular
delivery of nucleic acids both in vitro and in vivo
• Research products
– Develop novel research products including transfection reagents
and nucleic acid labeling kits
© 2007 Mirus Bio Corporation.
All rights reserved.
3
Presentation Overview
• Transfection– Introduction
– Methods of transfection
– Mechanism of lipid and/or polymer-mediated transfection
– Transfection optimization and critical parameters
• Applications of RNAi in the Study of Cell Biology– Introduction to RNA interference
– siRNA vs. miRNA. Roles and uses
– Profiling tissue miRNA
• Labeling miRNA & miRNA microarray
• Dynamic PolyConjugates– A New Synthetic Platform for Targeted in vivo Delivery of siRNA
• Summary
© 2007 Mirus Bio Corporation.
All rights reserved.
4
Nucleus
Plasmid DNA
NuclearEntry
CellularEntry
What is Transfection?
Cytoplasm
© 2007 Mirus Bio Corporation.
All rights reserved.
5
Nucleus
mRNA or viral RNA
CellularEntry
RNA Transfection
siRNA
No nuclear entry required
Cytoplasm
© 2007 Mirus Bio Corporation.
All rights reserved.
6
Transfection Methods
• Electroporation
– Delivery using high voltage, low amperage electric pulses
– Permeabilizes cell membrane to allow entry into the cell
• Calcium Phosphate Precipitation
– Addition of calcium and phosphate to DNA creates large precipitates
– Precipitates settle on cells and are taken up
• Lipid and/or Polymer-based Reagents
– Positively charged molecules that associate with nucleic acids
– Promote binding to cells and uptake by endocytosis
© 2007 Mirus Bio Corporation.
All rights reserved.
7
Electroporation
• Advantages
– Efficient method on many difficult to transfect cells and cell lines
– Delivers all types of nucleic acids
• Disadvantages
– Requirement
• large numbers of cells
• large amounts of nucleic acid
– Low cell survival
– Performed under serum-free conditions
– Requires expensive electroporation equipment and cuvettes
© 2007 Mirus Bio Corporation.
All rights reserved.
8
Calcium Phosphate Precipitation
• Advantages
– Reagents can be made in the lab
– Inexpensive
• Disadvantages
– Requires large amounts of nucleic acid
– Cumbersome protocols
– Finicky reagents
– Generally a low efficiency method
© 2007 Mirus Bio Corporation.
All rights reserved.
9
Lipid and/or Polymer-based Reagents
Advantages
– Simple to use
– High efficiency transfection
method
– Compatible with serum
containing media
Disadvantages
– Depending on the reagent
• Can exhibit cellular toxicity
• Can be expensive
© 2007 Mirus Bio Corporation.
All rights reserved.
10
Lipids Used in Transfection Reagents
• Two long alkyl chains attached to a polar head group
• Cationic lipids +/- a helper lipid
Polar
Head
Hydrophobic chains
© 2007 Mirus Bio Corporation.
All rights reserved.
11
Lipid Examples
H2NOPO O
-O O
O
O
( )7
)7( ( )7
( )7
O
( )7
)7( ( )7
( )7
N+ O
O
DOPE
(Polar, Helper)
DOTMA
(Cationic)
Lipofectin = 1:1 DOTMA:DOPE
© 2007 Mirus Bio Corporation.
All rights reserved.
12
Liposome Complexes
Cationic Lipid + Helper Lipid
Plasmid DNA
Liposome Multi-layered Liposome
Net (+) Charge
(DNA, Cationic + Helper Lipids)
(Not Drawn to Scale)
© 2007 Mirus Bio Corporation.
All rights reserved.
13
Cationic Lipid
Plasmid DNA
Lipoplexes
Net (+) Charge
Lipoplex Complexes
(Not Drawn to Scale)
© 2007 Mirus Bio Corporation.
All rights reserved.
14
Cationic Lipid
Cationic Polymer
Plasmid DNA
Lipopolyplex
(DNA, Lipid, Polycation)
Net (+) Charge
Polyplex
(DNA, Polycation)
Net (+) Charge
Different Types of ComplexesPolyplexes and Lipopolyplexes
(Not Drawn to Scale)
© 2007 Mirus Bio Corporation.
All rights reserved.
15
Nucleic acid
- ---
-
--
-- -REPULSION
Nucleus-
--
----
--
-
-
Cell
Charge Repulsion Between the Cell Surface and the
Nucleic Acid Inhibits Uptake
Cell Surface: First Transfection Barrier
© 2007 Mirus Bio Corporation.
All rights reserved.
16
- ---
-
--
-- -
+ Transfection Reagent
+
++
+
++
+
Nucleus
Endocytosis (Cellular Uptake)
----
-+
++
+
++
+
Condensation
Cell Association
--
Transfection Reagents Promote Cell Association and Uptake
+
++
+
++
+
Endosome
+
++
+
++
+
© 2007 Mirus Bio Corporation.
All rights reserved.
17
Nucleus
Endosomal Escape
Transfection Reagents Promote Endosomal Escape
+
++
+
++
+
+
++
+
++
+
Nuclear Entry
No evidence for promoting
nuclear entry
© 2007 Mirus Bio Corporation.
All rights reserved.
18
Visualization of the Transfection Process
1 hour 4 hours
8 hours 24 hours
Autofluorescing cells
Rhodamine-labeled DNA
Yellow Fluorescent Protein
COS-7 Cells
© 2007 Mirus Bio Corporation.
All rights reserved.
19
General Transfection Reagent Protocol
© 2007 Mirus Bio Corporation.
All rights reserved.
20
Transfection Optimization
• Amount of reagent and nucleic acid
• Complex formation time
• Cell confluency (density) at transfection
• Post-transfection incubation time
© 2007 Mirus Bio Corporation.
All rights reserved.
21
Transfection Optimization:DNA & Reagent Amount
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
COS-7 HEK 293CHO-K1
© 2007 Mirus Bio Corporation.
All rights reserved.
22
Transfection Optimization
• Amount of reagent and nucleic acid
• Complex formation time
• Cell confluency (density) at transfection
• Post-transfection incubation time
© 2007 Mirus Bio Corporation.
All rights reserved.
23
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)
Transfection Optimization:Complex Formation Time
CHO-K1 Cells - TransIT®-LT1 Reagent
0.E+107
4.0E+107
8.0E+107
1.2E+108
1.6E+108
2.0E+108
© 2007 Mirus Bio Corporation.
All rights reserved.
24
Transfection Optimization
• Amount of reagent and nucleic acid
• Complex formation time
• Cell confluency (density) at transfection
• Post-transfection incubation time
© 2007 Mirus Bio Corporation.
All rights reserved.
25
Transfection Optimization:Cell Confluency at 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
NIH3T3
Cells
© 2007 Mirus Bio Corporation.
All rights reserved.
26
Transfection Optimization
• Amount of reagent and nucleic acid
• Complex formation time
• Cell confluency (density) at transfection
• Post-transfection incubation time
© 2007 Mirus Bio Corporation.
All rights reserved.
27
Transfection Optimization:Post-transfection Incubation Time
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
3,500,000
0 6 12 18 24 30 36 42 48
Hours Post Transfection
Fir
efl
y L
ucif
erase E
xpressio
n (
RLU
s)
Firefly luc mRNA Delivered to NIH3T3 Cells using TransIT®-mRNA Kit
Lu
cif
era
se
Ac
tivit
y (
RL
Us)
© 2007 Mirus Bio Corporation.
All rights reserved.
28
Other Critical Parameters• Mycoplasma contamination
– Contamination is difficult to detect visually
– Can affect transfection results – decreased viability
• Use of endotoxin-free DNA
– Endotoxin can decrease cell viability and inhibit transfection
• Transfection of Cells at Consistent Passage Numbers
– Cells change during passage
– Changes can affect transfection results
• Transfection Comparisons to be done on the Same Day
– Transfection performance can vary day-to-day
© 2007 Mirus Bio Corporation.
All rights reserved.
29
Transfection Efficiency varies with Cell Line (e.g.TransIT®-LT1 Reagent)
© 2007 Mirus Bio Corporation.
All rights reserved.
30
Efficiency varies with transfection reagents
HeLa Cells Plated per 12-well (x 103)
Pe
rce
nt C
on
flu
en
cy (
line
s)
Lu
cife
rase A
ctivity (
RL
Us x
10
6)
(b
ars
)Transfection Reagent - 1 Reagent -2 Confluency at Transfection
0
1
2
3
4
5
6
7
25 50 75 100 125 150 200 250 300 350
0
20
40
60
80
100
© 2007 Mirus Bio Corporation.
All rights reserved.
31
0.00E+00
2.00E+07
4.00E+07
6.00E+07
8.00E+07
1.00E+08
1.20E+08
1.40E+08
3 4.5 6 3 4.5 6 10 15 20
TransIT-LT1 Reagent TransIT-293 Reagent PolyFect
Reagent Amount (ul) per 1.5 ug DNA
Lu
cif
era
se A
cti
vit
y (
RL
Us)
Transfection Results Vary Day-to-Day
Day 1 Transfections
Day 2 Transfections
1.4E+108
2.0E+107
4.0E+107
6.0E+107
8.0E+107
1.2E+108
1.0E+108
Lu
cif
era
se A
cti
vit
y (
RL
Us
)
0.E+107
Reagent-A Reagent-B Reagent-C
© 2007 Mirus Bio Corporation.
All rights reserved.
32
0.00E+00
2.00E+07
4.00E+07
6.00E+07
8.00E+07
1.00E+08
1.20E+08
1.40E+08
3 4.5 6 3 4.5 6 10 15 20
TransIT-LT1
Reagent
TransIT-293
Reagent
PolyFect
0.00E+00
1.00E+07
2.00E+07
3.00E+07
4.00E+07
5.00E+07
6.00E+07
3 4.5 6 3 4.5 6 10 15 20
TransIT-LT1
Reagent
TransIT-293
Reagent
PolyFect
Performance Trends Remain Similar
Day 1 Transfections Day 2 Transfections
1.4E+108
2.0E+107
4.0E+107
6.0E+107
8.0E+107
1.2E+108
1.0E+108
Lu
cif
era
se A
cti
vit
y (
RL
Us
)
0.E+107
Reagent Amount (µl) per 1.5 µg DNA
Reagent-A Reagent-B Reagent-C
Reagent Amount (µl) per 1.5 µg DNA
Reagent-A Reagent-B Reagent-C
1.0E+107
2.0E+107
3.0E+107
4.0E+107
6.0E+107
5.0E+108
0.E+107
© 2007 Mirus Bio Corporation.
All rights reserved.
33
Transfection Summary
• Transfection efficiency is highly dependent on cell line
• Optimize transfection efficiency by testing five key variables
– Reagent and DNA amount, complex formation time, confluency, and
post-transfection incubation time
• For Consistent Results
‒ Maintain similar passage numbers between experiments
– Perform critical comparisons in the same experiment
• Challenges
‒ Uniformity in efficiency
‒ Hard-to-transfect Cell lines