The overall goal of the following video is to demonstrate the use of a whole genome, short hairpin RNA or S-H-R-N-A library to rapidly conduct a positive selection screen. This is achieved by transduction of the target cells with 10 sub pools of the mission, lenti plex, pooled whole genome, S-H-R-N-A library, followed by addition of the therapeutic component. As a second step, the HRNA template material is recovered, which enables the deconvolution of the pooled screen.
The recovered S-H-R-N-A is then cloned into the vector and transformed into competent bacteria. Next plasmid DNA samples are sequenced and S-H-R-N-A targets are identified using the lenti PLX, HRNA sequence search database. The results of the experiment performed in this video reveal the genes involved in the cytotoxicity of an epithelial tumor cell line when exposed to anti-tumor pharmacological agents based on the number of sequencing hits The mission.
Lin Duplex pooled. HRNA library contains the RNAI consortium or TRC collection in 10 matrix tubes and can be used to rapidly perform high throughput loss of gene function studies. In this study, it is used to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of a 5 49 cells when exposed to trail and paclitaxel Prior to transduction of target cells.
CED the appropriate number of 60 millimeter dishes with enough cells to obtain approximately 70%co fluency on the following day. Incubate cells at 37 degrees Celsius overnight. Growth rates will vary by cell type and must be determined before starting the screen to prepare for transduction of cells with the mission lenti plex pooled, S-H-R-N-A library thaw the lentiviral particles on ice, transfer the thawed particles to a laminar flow hood, keeping on ice if not being used immediately.
Calculate how much of each individual viral subpool to add to the cells to get a multiplicity of infection or MOI of one across all cell culture dishes. A low MOI is used to reduce the probability of multiple integrins per cell, dilute the calculated amount of virus in 100 to 300 microliters of complete media in order to ensure better distribution of the virus when added to the cell culture dish, then remove media from the preceded cells. Add complete media containing poly brain solution to each dish to the media.
Add HRNA lentiviral particles to the appropriate dishes in accordance with the predetermined experimental design. Gently swirl the plates to evenly distribute the virus across cells. Incubate 18 to 20 hours at 37 degrees Celsius in a humidified incubator in an atmosphere of 5%carbon dioxide.
The next day, aspirate the virus containing media and add fresh complete media without poly brain. Incubate the cells at 37 degrees Celsius overnight on the fourth day, aspirate the media from the cells and add fresh media containing the therapeutic component at a predetermined optimal concentration. Then treat the cells with the component for a predetermined duration.
In this study, cells are treated with 100 nanograms per milliliter of trail and one micromolar of paclitaxel for 48 hours. Following treatment, surviving cells should be reseeded into T 75 flasks and allowed to expand until nearly 100%confluent to perform polyclonal deconvolution. The heterogeneous population of cells is harvested from each subpool and the genomic DNA is isolated.
Begin the S-H-R-N-A template recovery procedure with setup of the PCR reaction as described in the written protocol. Amplification primers at a concentration of 20 millimolar are included with the lenti plex kit. Prepare a positive control using the mission SHRA human positive control vector diluted to the appropriate concentration as a template.
Successful amplification with this template indicates that the PCR assay components and cycling conditions are adequate. Save the PCR program listed in the written protocol into the thermocycler and perform target amplification. Once the cycle is complete, combine three microliters of each PCR reaction with one microliter of dye and electrophoresis alongside a 100 base pair low ladder on a 1%aros gel to confirm the presence of a 309 base pair amplicon.
At this point, the PCR Amplicons are sub cloned Topo clone the PCR products using the topo ta cloning kit following the manufacturer's protocol. The result is that one PCR product is contained in each vector. Once the sub cloning is complete, isolate 250 individual bacterial colonies, each of which contains an individual PCR amplicon.
Grow the individual colonies in two milliliter cultures from which the plasmid DNA can be extracted using the Gen Elute plasmid mini prep kit following digestion of the purified plasmid DNA with echo R one, the presence of the insert can be confirmed by electrophoresis. Finally, from the sequenced plasma DNA samples, the S-H-R-N-A insert can be identified using the Plex HRNA sequence search database provided with the PLX library. Find the search box on the enclosed S-H-R-N-A sequence search access database form to the beginning of the HRNA sequence.
Enter the next 10 to 21 nucleotides of the query sequence. Then select the species of the pooled S-H-R-N-A. Optionally select the sub pool from which the sequence was found.
Now click on the find potential hits button to perform the search. This should identify the corresponding T-R-C-S-H-R-N-A sequences that match the sequencing data. Sigma aldrich's, your favorite gene website can then be used to find more information on the TR CS HRA sequences identified and the corresponding gene targets identified target genes should be validated by repeating the original screening assay with the original TRC clone plus at least two additional RNAs that target the same gene using a one HRNA to one 60 millimeter dish format.
True hits will display the same phenotype in a majority of dishes. Clones containing the cloning vector with one individual PCR fragment were isolated using the SHRA template recovery procedure and the HRNA insert was identified using the lenti plex SHRA sequence search database. 250 clones were sequenced.
Of these 25 were represented by multiple clones, and the remaining 225 were represented by only one clone and not pursued. As shown here, several candidate genes, including TBX three, PPP two, ca, and AKT two were represented by multiple clones. The T box transcription factor TBX three appeared in a total of four clones and has been implicated in tumor migration.
PPP two ca downregulation has been demonstrated to maintain the growth of ln cap cells cultured in androgen deficient conditions by relieving the androgen deprivation induced cell cycle arrest and preventing apoptosis. Furthermore, AKT two is Arak Beta Syrian three Anine protein kinase and putative oncogene demonstrated to play several roles in cancer development. After watching this video, you should have a good understanding of how to use the mission.
Len Plex pooled SH RNA library to rapidly conduct high throughput loss of gene function studies.
