慢病毒包装教程(Lentivirus Packaging and Production)

Lentivirus Packaging and Production

The laboratories of Didier Trono (EPFL) and Robert Weinberg (Whitehead Institute)

have deposited plasmids for the production of lentiviral particles. These plasmids can

be used with many lentiviral vectors, including The RNAi Consortium shRNA vectors

being distributed by Sigma (i.e. MISSION shRNAs) and Open Biosystems (i.e. TRC

shRNAs).

Overview

For producing lentiviral particles, you typically need three components: 1) a lentiviral

vector, such as

pLKO.1

or

pLVTHM

, containing the shRNA or transgene, 2) a

packaging vector, such as

psPAX2

or

pCMV-dR8.2 dvpr

, and 3) an envelope vector,

such as

pMD2.G

or

pCMV- VSVG

.

For most applications, you can produce viral particles by transient transfection of

293T cells with a 2nd generation packaging system (e.g. packaging plasmid psPAX2

and envelope plasmid pMD2.G).

2nd Generation Packaging System

In general, lentiviral vectors with a wildtype 5' LTR need the 2nd generation packaging

system because these vectors require TAT for activation. All lentiviral vectors from the

Trono or Aebischer lab require packaging with a 2nd generation system.

Below are two 2nd generation systems. Lentiviral plasmids based on pLKO.1 can be

packaged with either system, although the first system has been reported to produce

higher titer. See

Addgene's pLKO.1 Protocol

for producing lentiviral particles.

2nd generation system deposited by the Trono lab:

ID

Plasmid

Description

td>

12260

psPAX2

2nd

generation

packaging

plasmid

for

producing

viral

particles.

psPAX2

contains

a

robust

CAG

promoter

for

efficient

expression

of

packaging

proteins.

Trono

lab

and

Aebischer

lab

lentiviral

vectors

require

psPAX2.

Produces

higher titer than pCMV-dR8.2 dvpr.

pMD2.G

Envelope plasmid for producing viral particles

12259

2nd generation system deposited by the Weinberg lab:

ID

td>

8455

Plasmid

Description

pCMV-dR8.2 dvpr

2nd

generation

packaging

plasmid

for

producing

viral particles

pCMV-VSVG

Envelope plasmid for producing viral particles

8454

3rd Generation Packaging System

The 3rd generation packaging system offers maximal biosafety but is more

cumbersome to use, as it involves the transfection of four different plasmids in the

producer cells (two packaging plasmids, an envelope plasmid, and the lentiviral

vector).

If you wish to use this system, you need to have a lentiviral vector with a chimeric 5'

LTR in which the HIV promoter is replaced with CMV or RSV, thus making it

TAT-independent. Examples of these vectors include pLKO.1, pLL3.7, pLB, pLenti6,

pSico, pCL, and pCS. Most Aebischer and Trono Lab lentiviral vectors CANNOT be

used with this system. A lentiviral vector carrying a chimeric 5' LTR can be packaged

with either the 2nd or 3rd generation packaging system.

ID

td>

12251

Plasmid

Description

pMDLg/pRRE

3rd generation packaging plasmid for producing viral

particles

pRSV- Rev

3rd generation packaging plasmid for producing viral

particles

Envelope plasmid for producing viral particles

12253

12259

pMD2.G

More information

?

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Click

here

to browse other RNAi vectors, or search for plasmids using the

search bar at the top of the page.

Trono Lab website

or

Lentiweb

: information and a discussion forum on cloning,

packaging, and other protocols.

Moffat J et. al. 2006. A lentiviral RNAi library for human and mouse genes

applied to an arrayed viral high-content screen. Cell 124:1283-1298.

(

PubMed

)

Ventura et. al. 2004. Cre- lox-regulated conditional RNA interference from

transgenes. PNAS 2004 Jul 13;101(28):10380-5. (

PubMed

)

Naldini L et. al. 1996. In vivo gene delivery and stable transduction of

nondividing cells by a lentiviral vector. Science 272:263-267. (

PubMed

)

Dull et al., A Third-Generation Lentivirus Vector with a Conditional Packaging

System. J. Virol. 1998 72(11): 8463-8472. (

PubMed

)

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Zufferey R et. al. 1997. Multiply attenuated lentiviral vector achieves efficient

gene delivery in vivo. Nat Biotechnol 15(9):871-5. (

PubMed

)

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Zufferey R et. al. 1998. Self- inactivating lentivirus vector for safe and efficient

in vivo gene delivery. J Virol 72(12):9873-80. (

PubMed

)

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Cell Line

The 293T cell line for producing lentiviral particles can be obtained from

GenHunter

.

pLKO.1 Protocol

pLKO.1 - TRC Cloning Vector

Addgene Plasmid 10878. Protocol Version 1.0. December 2006.

Copyright Addgene 2006, All Rights Reserved. This protocol is provided for your convenience. See

warranty information

in appendix.

Click

here

for a printable copy.

Table of Contents

A. pLKO.1-TRC Cloning Vector

o

A.1 The RNAi Consortium

o

A.2 Map of pLKO.1

o

A.3 Related plasmids

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B. Designing shRNA Oligos for pLKO.1

o

B.1 Determine the optimal 21-mer targets in your gene

?

o

B.2 Order oligos compatible with pLKO.1

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C. Cloning shRNA oligos into pLKO.1

o

C.1 Recommended materials

o

C.2 Annealing oligos

o

C.3 Digesting pLKO.1 TRC-Cloning Vector

o

C.4 Ligating and transforming into bacteria

D. Screening for Inserts

o

D.1 Recommended materials

o

D.2 Screening for inserts

E. Producing Lentiviral Particles

o

E.1 Recommended materials

o

E.2 Protocol for producing lentiviral particles

F. Infecting Target Cells

o

F.1 Recommended materials

o

F.2 Determining the optimal puromycin concentration

o

F.3 Protocol for lentiviral infection and selection

G. Safety

H. References

o

H.1 Published articles

o

H.2 Web resources

I. Appendix

o

I.1 Sequence of pLKO.1 TRC-Cloning Vector

o

I.2 Recipes

o

I.3 Warranty information

Back to Top

A. pLKO.1-TRC Cloning Vector

A.1 The RNAi Consortium

The

pLKO.1

cloning vector is the backbone upon which

The RNAi Consortium (TRC)

has built a library of shRNAs directed against 15,000 human and 15,000 mouse

genes. Addgene is working with the TRC to make this shRNA cloning vector available

to the scientific community. Please cite

Moffat et al., Cell 2006 Mar; 124(6):1283-98

(

PubMed

) in all publications arising from the use of this vector.

A.2 Map of pLKO.1

pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for

expression of shRNAs. pLKO.1 can be introduced into cells via direct transfection, or

can be converted into lentiviral particles for subsequent infection of a target cell line.

Once introduced, the puromycin resistance marker encoded in pLKO.1 allows for

convenient stable selection.

Figure 1 : Map of pLKO.1 containing an shRNA insert. The original

pLKO.1-TRC cloning vector has a 1.9kb stuffer that is released by

digestion with AgeI and EcoRI. shRNA oligos are cloned into the AgeI

and EcoRI sites in place of the stuffer. The AgeI site is destroyed in

most cases (depending on the target sequence), while the EcoRI site is

preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer,

visit

/10878

.

Description

Vector Element

U6

cPPT

Human U6 promoter drives RNA Polymerase III transcription for

generation of shRNA transcripts.

Central polypurine tract, cPPT, improves transduction efficiency by

facilitating nuclear import of the vector's preintegration complex in the

transduced cells.

Human phosphoglycerate kinase promoter drives expression of

puromycin.

Puromycin resistance gene for selection of pLKO.1 plasmid in

mammalian cells.

3' Self- inactivating long terminal repeat.

f1 bacterial origin of replication.

Ampicillin resistance gene for selection of pLKO.1 plasmid in bacterial

cells

pUC bacterial origin of replication.

5' long terminal repeat.

Rev response element.

hPGK

Puro R

sin 3'LTR

f1 ori

Amp R

pUC ori

5'LTR

RRE

Figure 2 : Detail of shRNA insert. The U6 promoter directs RNA

Polymerase III transcription of the shRNA. The shRNA contains 21

containing an XhoI restriction site, and 21

are complementary to the

by a polyT termination sequence for RNA Polymerase III.

A.3 Related Products

The following plasmids available from Addgene are recommended for use in

conjunction with the pLKO.1 TRC-cloning vector.

Plasmid (Addgene ID #)

Description

pLKO.1 - TRC control (10879)

Negative control vector containing non-hairpin

insert.

pLKO.1 - scramble shRNA

(1864)

psPAX2 (12260)

pMD2.G (12259)

Negative control vector containing scrambled

shRNA.

Packaging plasmid for producing viral particles.

Envelope plasmid for producing viral particles.

Note: pLKO.1 can also be used with packaging plasmid

pCMV-dR8.2 dvpr (Addgene

#8455)

and envelope plasmid

pCMV-VSVG (Addgene #8454)

from Robert

Weinberg's lab. For more information, visit Addgene's

Mammalian RNAi Tools

page.

Several other laboratories have deposited pLKO derived vectors that may also be

useful for your experiment. To see these vectors, visit Addgene's website and

search

for

.

Back to Top

B. Designing shRNA Oligos for pLKO.1

B.1 Determining the Optimal 21-mer Targets in your Gene

Selection of suitable 21-mer targets in your gene is the first step toward efficient gene

silencing. Methods for target selection are continuously being improved. Below are

suggestions for target selection.

1. Use an siRNA selection tool to determine a set of top-scoring targets for your gene.

For example, the Whitehead Institute for Biomedical Research hosts an siRNA

Selection Program that can be accessed after a free registration

(

/bioc/siRNAext /

). If you have MacOS X, another excellent

program is iRNAi, which is provided free by the company Mekentosj

(

/irnai/

).

A summary of guidelines for designing siRNAs with effective gene silencing is

included here:

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Starting at 25nt downstream of the start codon (ATG), search for 21nt

sequences that match the pattern AA(N

19

). If no suitable match is found,

search for NAR(N

17

)YNN, where N is any nucleotide, R is a purine (A,G), and

Y is a pyrimidine (C,U).

G-C content should be 36-52%.

Sense 3' end should have low stability

–

at least one A or T between position

15-19.

Avoid targeting introns.

Avoid stretches of 4 or more nucleotide repeats, especially repeated Ts

because polyT is a termination signal for RNA polymerase III.

2. To minimize degradation of off-target mRNAs, use NCBI's BLAST program. Select

sequences that have at least 3 nucleotide mismatches to all unrelated genes.

Addgene recommends that you select multiple target sequences for each

gene. Some sequences will be more effective than others. In addition,

demonstrating that two different shRNAs that target the same gene can

produce the same phenotype will alleviate concerns about off-target effects.

B.2 Ordering Oligos Compatible with pLKO.1

To generate oligos for cloning into pLKO.1, insert your sense and antisense

sequences from step B.1 into the oligos below. Do not change the ends; these bases

are important for cloning the oligos into the pLKO.1 TRC-cloning vector.

Forward oligo:

5' CCGG

—

21bp sens e

—

CTCGAG

—

< br>21bp antisense

—

TTTTTG 3'

Reverse oligo:

5' AATTCAAAAA

—

21bp sense

—

CTCGAG

—

21bp antisense 3'

For example, if the target sequence is (AA)TGCCTACGTTAAGCTATAC, the oligos

would be:

Forward oligo:

5'

CCGG

AATGCCTACG TTAAGCTATAC

CTCGAG

GTATAGCTTAA CGTAGGCATT

TTT

TTG 3'

Reverse oligo:

5'

AATTCAAAAA

AATG CCTACGTTAAGCTATAC

CTCGAG

GTATA GCTTAACGTAGGC

ATT

3'

Back to Top

C. Cloning Oligos into pLKO.1

The pLKO.1-TRC cloning vector contains a 1.9kb stuffer that is released upon

digestion with EcoRI and AgeI.

The oligos from section B contain the shRNA sequence flanked by sequences that

are compatible with the sticky ends of EcoRI and AgeI. Forward and reverse oligos

are annealed and ligated into the pLKO.1 vector, producing a final plasmid that

expresses the shRNA of interest.

C.1 Recommended Materials

Material

AgeI

EcoRI

T4 DNA ligase

NEB buffer 2

Vendor and catalog #

New England Biolabs (NEB) #R0552S

NEB #R0101S

NEB #M0202S

NEB #B7002S

DH5 alpha competent cells

I

nvitrogen #18258-012

Qiaquick gel extraction kit

Qiagen #28704

Low melting point agarose

Sigma #A9414

Luria Broth Agar (LB agar)

American Bioanalytical: #AB01200-02000

Ampicillin

Carbenicillin

C.2 Annealing Oligos

1. Resuspend oligos in ddH

2

O to a concentration of 20 μM, then mix:

5 μL

Forward oligo

5 μL

Reverse oligo

5 μL

10x NEB buffer 2

35 μL

ddH

2

O

VWR: #7177-48-

2. Use at 100 μg/mL.

VWR: #80030-

956. Use at 100 μg/mL.

2. Incubate for 4 minutes at 95

o

C in a PCR machine or in a beaker of boiling water.

3. If using a PCR machine, incubate the sample at 70

o

C for 10 minutes then slowly

cool to room temperature over the period of several hours. If using a beaker of water,

remove the beaker from the flame, and allow the water to cool to room temperature.

This will take a few hours, but it is important for the cooling to occur slowly for the

oligos to anneal.

C.3 Digesting pLKO.1 TRC Cloning Vector

1. Digest pLKO.1 TRC-cloning vector with AgeI. Mix:

6 μg

5 μL

1 μL

pLKO.1 TRC- cloning vector (maxiprep or miniprep DNA)

10x NEB buffer 1

AgeI

to 50 μL

ddH

2

O

>

Incubate at 37

o

C for 2 hours.

2. Purify with Qiaquick gel extraction kit. Elute in 30 μL of ddH

2

O.

3. Digest eluate with EcoRI. Mix:

30 μL

pLKO.1 TRC-cloning vector digested with AgeI

5 μL

10x NEB buffer for EcoRI

1 μL

EcoRI

14 μL

ddH

2

O

>

Incubate at 37

o

C for 2 hours.

4. Run digested DNA on 0.8% low melting point agarose gel until you can distinctly

see 2 bands, one 7kb and one 1.9kb. Cut out the 7kb band and place in a sterile

microcentrifuge tube.

When visualizing DNA fragments to be used for ligation, use only

long- wavelength UV light. Short wavelength UV light will increase the chance

of damaging the DNA.

5.

Purify the DNA using a Qiaquick gel extraction kit. Elute in 30 μL of ddH

2

O.

6. Measure the DNA concentration.

C.4 Ligating and Transforming into Bacteria