pLKOshRNA病毒质粒载体构建protocol方法详解

干扰shRNA病毒质粒构建

Plasmid Cart

Your cart is empty.

Search for Plasmids: Recently Viewed

pLKO.1 - TRC clo...

Plasmid 10878

Information

Browse

Search

Pricing

FAQ Plasmid 12247 Mammalian RNAi T... Collection pLVTHM

> 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 in appendix.

Click for a printable copy.

Table of Contents

o A.1 The RNAi Consortium

o A.2 Map of pLKO.1

o A.3 Related plasmids

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

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

o D.1 Recommended materials

干扰shRNA病毒质粒构建

o D.2 Screening for inserts

o E.1 Recommended materials o E.2 Protocol for producing lentiviral particles o F.1 Recommended materials o F.2 Determining the optimal puromycin concentration o F.3 Protocol for lentiviral infection and selection o H.1 Published articles o H.2 Web resources

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 cloning vector is the backbone upon which 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.

干扰shRNA病毒质粒构建

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 Human U6 promoter drives RNA Polymerase III transcription for generation of shRNA transcripts. Central polypurine tract, cPPT, improves transduction effi

ciency 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.

cPPT

hPGK

Puro R

sin 3'LTR

干扰shRNA病毒质粒构建

A.3 Related Products

The following plasmids available from Addgene are recommended for use in conjunction with the pLKO.1 TRC-cloning vector.

干扰shRNA病毒质粒构建

Note: pLKO.1 can also be used with packaging plasmid and envelope plasmid from Robert Weinberg's lab. For more information, visit Addgene's 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 .

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 (). If you have MacOS X, another excellent program is iRNAi, which is provided free by the company Mekentosj ().

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

Starting at 25nt downstream of the start codon (ATG), search for 21nt sequences that match the pattern AA(N19). If no suitable match is found, search for NAR(N17)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.

干扰shRNA病毒质粒构建

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 …… 此处隐藏：5338字，全部文档内容请下载后查看。喜欢就下载吧 ……