pLKOshRNA病毒质粒载体构建protocol方法详解(3)

b. The target cells should be approximately 80-90% confluent. c. Dilute puromycin in the preferred culture media for your target cells. The final concentration of puromycin should be from 1-10 μg/mL in 1 μg/mL increments.

d. Label plates from 1-10 and add appropriate

puromycin-containing media to cells.

e. Examine cells each day and change to fresh

puromycin-containing media every other day.

f. The minimum concentration of puromycin that results in

complete cell death after 3-5 days is the concentration that should be used for selection in your experiments. (You may wish to

repeat this titration with finer increments of puromycin to

determine a more precise optimal puromycin concentration.)

F.3. Protocol for Lentiviral Infection and Selection

a. Plate target cells and incubate at 37oC, 5% CO2 overnight.

b. Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg/mL polybrene.

Polybrene increases the efficiency of viral infection.

干扰shRNA病毒质粒构建

However, polybrene is toxic to some cell lines. In these cell

lines, substitute protamine sulfate for polybrene.

c. Add lentiviral particle solution from step E. For a 6 cm target plate, add between 0.05-1 mL virus (add ≥0.5 mL for a high MOI, and ≤0.1 mL for a low MOI). Scale the amount of virus added

depending on the size of your target plate.

MOI (multiplicity of infection) refers to the number of

infecting viral particles per cell. Addgene recommends that

you test a range of MOIs to determine the optimal MOI for

infection and gene silencing in your target cell line.

d. Incubate cells at 37oC, 5% CO2 overnight. Day 3:

e. Change to fresh media 24 hours after infection.

If viral toxicity is observed in your cell line, you may

decrease the infection time to between 4 - 20 hours.

Remove the virus-containing media and replace with fresh

media. Do not add puromycin until at least 24 hours after

infection to allow for sufficient expression of the puromycin

resistance gene.

f. To select for infected cells, add puromycin to the media at the concentration determined in step E.2.

Addgene recommends that you maintain one uninfected

plate of cells in parallel. This plate will serve as a positive

control for the puromycin selection.

g. Change to fresh puromycin-containing media as needed every few days.

h. Assay infected cells. The following recommendations are

guidelines for the number of days you should wait until harvesting your cells. However, you should optimize the time based on your cell line and assay: Protein knockdown (western blot) ≥ 4 days mRNA knockdown (quantitative PCR) ≥ 3 days

干扰shRNA病毒质粒构建

Phenotypic assay

≥ 4 days G. Safety

BL2 safety practices should be followed when preparing and handling lentiviral particles. Personal protective clothing should be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should be decontaminated with at least 10% bleach. Laboratory materials that come in contact with viral particles should be treated as biohazardous waste and autoclaved. Please follow all safety guidelines from your institution and from the CDC and NIH for work in a BL2 facility. If you have any questions about what safety practice to follow, please contact your institution's safety office.

To obtain the MSDS for this product, visit /sitemap and follow the MSDS link.

H. References

H.1. Published Articles

Khvorova A et. al. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209-216. (PubMed)

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. ()

Naldini L et. al. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267. () Schwarz DS et. al. 2003. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199-208. ()

Stewart SA et. al. 2003. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9(4):493-501. (PubMed)

Zufferey R et. al. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15(9):871-5. ()

干扰shRNA病毒质粒构建

Zufferey R et. al. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873-80. () H.2. Web resources

Addgene's mammalian RNAi website: The RNAi Consortium (TRC): www.broad.mit.edu/genome_bio/trc/rnai.html

Background on RNAi mechanism: Whitehead siRNA Selection Program: Mekentosj iRNAi Program:

I. Appendix

I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here (/10878) to see the sequence of

pLKO.1 TRC-cloning vector. The vector is 8901 base pairs total, and the stuffer insert is shown in all capital letters.

I.2. Recipes Luria Broth Agar (LB agar) + antibiotic

Per 40 grams of powder from American Bioanalytical catalog # AB01200-02000, LB contains:

10g tryptone

5g yeast extract

10g sodium chloride

15g agar

> Prepare LB agar solution by dissolving 40g of LB powder in 1L of distilled water. Autoclave and cool to 55oC. Add 1mL of

100mg/mL ampicillin or carbenicillin to obtain a final concentration of 100 μg/mL antibiotic. Pour plates and store at 4oC.

干扰shRNA病毒质粒构建

Prepare a 1mg/mL solution of polybrene (Sigma-Aldrich catalog #H9268) in 0.9% NaCl. Autoclave to sterilize. Stock solution is stable at 4oC for up to one year. The powder form of polybrene is stable at 4oC for several years.

Store protamine sulfate (MP Biomedicals catalog #194729) at 4oC. Freel …… 此处隐藏：4877字，全部文档内容请下载后查看。喜欢就下载吧 ……