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pGPU6/GFP/Neo plasmid: Features

Nucleotide position

CMV IE promoter

1-589

GFP ORF

613-1410

SV40 polyA

1499-1549

T3 promoter binding site

1602-1621

HU6 promoter

1720-1992

T7 promoter binding site

2005-2026

F1 origin

2033-2488

SV40 promoter

2829-3011

Kanamycin/Neomycin resistance gene

3013-3807

pUC origin of replication

4392-5035

F-07

Component

20 μl

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pGPU6/GFP/Neo (circular) (50 ng/ul)

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20μl

pGPU6/GFP/Neo (linearized) (50 ng/ul)

10 μl

10 μl

pGPU6/GFP/Neo Negative control (0.5 μg/μl)

10 μl

10 μl

GAPDH Control Insert (20 nM)

0.5 ml

0.5 ml

10×shDNA Annealing Solution

Other Required Materials

Ligation and transformation

? shRNA coding inserts: Two complementary carefully designed oligonucleotides targeting the gene of interest for RNAi
? DNA ligase, ligase reaction buffer, and competent?E. coli?cells are needed to subclone the shRNA inserts.
? Kanamycin containing plates and liquid media will also be needed to propagate the plasmids.

Plasmid purification

For efficient transfection into mammalian cells it is crucial that preparations of pGPU6/GFP/Neo be very pure.

Mammalian cell transfection reagents

The optimal mammalian cell transfection conditions including transfection agent and plasmid amount must be determined empirically.

Cell culture facility and supplies

In addition to routine cell culture media, culture media containing G418 (a neomycin analog) will be needed for selection of pGPU6/GFP/Neo-transfected cells.

Optimizing Antibiotic Selection Conditions

G418 titration (kill curve)

a.?????? Plate 20,000 cells into each well of a 24 well dish containing 1 ml of culture medium.
b.?????? After 24 hr, add 500 μl culture medium containing 25–4000 μg/ml G418.
c.?????? Culture the cells for 10–14 days, replacing the antibiotic-containing medium every 3 days.
d.?????? Examine the dishes for viable cells every 2 days.
Identify the lowest G418 concentration that begins to give massive cell death in approximately 7–9 days, and kills all cells within 2 weeks. Use this G418 concentration to select cells containing the pGPU6/GFP/Neo plasmid after transfection.

Optimal plating density

a.?????? Plate several different amounts of cells into separate wells of a 24 well dish containing 1 ml of culture medium.
b.?????? After 24 hr, add 500 μl culture medium containing G418; use the concentration identified in the previous experiment.
c.?????? Culture the cells for 5–14 days, replacing the antibiotic-containing medium every 3 days.
Identify the cell plating density that allows the cells to reach 80% confluency before massive cell death begins; and use it to plate cells transfected with your pGPU6/GFP/Neo clone.

Cloning shRNA Inserts into?pGPU6/GFP/Neo

Prepare a 1 μg/μl solution of each oligonucleotide

a.?????? Dissolve the shRNA template oligonucleotides in approximately 100 μl of nuclease-free water.
b.?????? Dilute 1 μl of each oligonucleotide 1:100 to 1:1000 in TE (10 mM Tris, 1 mM EDTA) and determine the absorbance at 260 nm. Calculate the concentration (in μg/ml) of the shRNA oligonucleotides by multiplying the A260 by the dilution factor and then by the extinction coefficient (~33 μg/ml).
Dilute the oligonucleotides to approximately 100 μM.

Anneal the shRNA template oligonucleotides

a.?????? Assemble the 50 μl annealing mixture as follows:

b.?????? Heat the mixture to 95°C for 3 min, then turn off the heater and cool to room temperature slowly.
c.?????? The annealed shRNA template insert can either be ligated into a pGPU6/GFP/Neo vector or stored at –20°C for future ligation.

Transfecting pGPU6/GFP/Neo into Mammalian Cells

Transfect cells and culture 24 hr without selection

We recommend using GenePharma’s RNAi-Mate transfection reagent (Cat. No. C-01) to deliver pGPU6/GFP/Neo plasmids into mammalian cells with high efficiency and minimal toxicity. Follow the instructions for using RNAi-Mate provided with the product. RNAi-Mate is a proprietary formulation of polyamines that can be used in the presence or absence of serum in the culture medium. It is suitable for the transfection of a wide variety of cell types.
Transfect the purified plasmid into the desired cell line, plate transfected cells at the optimal plating density identified earlier, and culture for 24 hr without selection.
It is important to include two non-transfected control cultures. One is subjected to neomycin or G418 selection to control for the fraction of cells that survive selection; it will help determine the effectiveness of the transfection and selection. The second control is grown without neomycin or G418 selection as a positive control for cell viability.

Selecting Antibiotic-Resistant Transfected Cells

Once they are prepared, pGPU6/GFP/Neo shRNA expression vectors can be used in transient shRNA expression assays, or to create cell populations or a clonal cell line that stably expresses your shRNA. Note that with normal (non-transformed) and primary cell lines, it may be difficult to obtain clones that stably express shRNA. For these types of cells, we recommend choosing the antibiotic selection strategies outlined in sections 1 and 2 below.

Short term antibiotic selection for enrichment of cells that transiently express the shRNA

In experiments where the transfection efficiency is low, a rapid antibiotic selection can be used to kill cells that were not transfected with the pGPU6/GFP/Neo shRNA expression vector. This enrichment for transfected cells can be useful for reducing background when analyzing gene knockdown.
a.?????? Culture the cells for 1–3 days in the antibiotic-containing medium to enrich the culture for cells that were successfully transfected.
Analyze the population for an expected phenotype and/or the expression of the target gene.

Selecting a population of cells that stably express the shRNA

Creating a population of cells stably expressing the shRNA involves treating cells with neomycin or G418 for several days to eliminate cells that were not transfected. The surviving cell population can then be maintained and assessed for reduction of target gene expression.
a.?????? Culture the cells in medium containing neomycin or G418 until all of the cells in the non-transfected control culture are killed. At this point, the selection is complete and the cells can be grown without antibiotic until they repopulate the culture vessel.
b.?????? Analyze expression of the target gene at any time after the cells in the non-transfected control culture have been killed.
Pool and passage antibiotic-resistant cell cultures as needed. It is a good idea to periodically grow the cells with a minimal level of antibiotic selection, to prevent the accumulation of cells that no longer express antibiotic resistance. Often this “minimal level” is about half the antibiotic concentration used to kill off non-transfected cells, but this value varies widely among different cell types.

Selecting for clones that stably express the shRNA

For many researchers, the goal is to create a clonal cell line that expresses the shRNA template introduced with pGPU6/GFP/Neo. Cloning stably expressing cell lines is advantageous because strains that exhibit the desired amount of gene knockdown can be identified and maintained, and clones that are neomycin-resistant but which do not express the shRNA can be eliminated.
Typically, the levels of shRNA expression and gene knockdown vary widely among cells. In fact, pGPU6/GFP/Neo-transfected cells that survive antibiotic selection may not have a significant reduction in expression of the target gene. Instead, they may have found a way to mitigate the effects of a reduction in the target gene expression by compensating in another fashion or by shutting down expression of the shRNA. To avoid this, it can be useful to isolate clones that can be screened to identify the cells that cause the desired reduction in target gene expression.

Troubleshooting

Poor ligation?efficiency

If the ligation reaction is inefficient, then there will be relatively few plasmids to transform. Possible causes of poor ligation efficiency include:
a.????The concentration of the annealed shRNA template insert is lower than expected.
Evaluate ~5 μl of the insert DNA using a 12% native polyacrylamide gel and compare its ethidium bromide staining to bands from a molecular weight marker or another standard of known concentration.
b.????The ligase or ligase reaction buffer have become inactive.
Test your ligation components using another vector and insert or replace your ligation components and retry the shRNA insert cloning.
c.????One or both of the hairpin shRNA template oligonucleotides have high levels of non-full-length products.
The size of oligonucleotides can be evaluated on an 12% native polyacrylamide gel.
d.????The oligonucleotide annealing reaction was ineffective.
A low concentration of one of the oligonucleotides or incomplete denaturation of individual oligonucleotides could have reduced the relative amount of dsDNAs.
Compare the annealed shRNA template insert to each of the single-stranded oligonucleotides using native 8–12% polyacrylamide gel electrophoresis. If the annealed shRNA template insert has bands corresponding to the single-stranded oligonucleotides, then adjusting the concentrations of the single-stranded DNA molecules and heat-denaturing at a higher temperature during shRNA insert preparation might improve the percentage of dsDNA products. Alternatively, in some cases, gel purifying the band corresponding to annealed insert may result in better ligation.