The overall goal of the following experiment is to conduct a high throughput genome wide screen of micro RNA targets using the target ID library. This is achieved through cell transfection with the library plasmid. A genome-wide collection of cloned CDNAs transfection is followed by selection for stable cell lines.
The established library cells are then transfected with the micro RNA expression construct to allow co-expression of the micro RNA target and thymidine kinase. At this point, ganciclovir is used to select for cells expressing specific micro RNA targets by selecting against thymidine kinase expression. The CD NA containing micro RNA target sites are then PCR amplified using the target ID amplification primers in order to sequence and identify the micro RNA library targets.
Ultimately, results are obtained that identify specific micro RNA targets in cells based on deep sequencing analysis. The mission target ID library is designed to assist in the discovery and identification of gene targets for micro RNA. Here we demonstrate its use and application.
Prior to the start of this protocol, choose a cell line that does not express or expresses low levels of the IRA of interest culture and expand the cells to obtain approximately two times 10 to the seven cells at 80%co fluency. After trypsin, the cells transfer them to a 15 milliliter sterile screw top tube pellet the trypsin cells at 200 times G for five minutes. Following removal of the medium resus, suspend the cell pellet to wash, centrifuge the cells again and repeat the wash a second time.
Meanwhile, prepare enough 0.5 milliliter tubes for the number of transfect by adding two micrograms of the target ID library to each tube. Now resuspend the cell pellet within the 15 milliliter tube using 600 microliters of a maxim Nucle affection solution per transfection. One reaction at a time.
Add 100 microliters of cells to the aliquot, two micrograms of plasmid mixing with the pipette tip. Transfer each mixture to a nuclear effector vete. Insert one vete into the nuclear effector instrument and run an optimized program appropriate for the cell line.
Next, fill a transfer pipette with prewarm medium. Take up the transfected cells in the same pipette and transfer to a single well of a previously prepared six Well plate. Repeat this process for each NU nuclear affection following NU nuclear affection.
Return the cells to the growth chamber for overnight incubation the next day. Replace the medium and allow the cells to recover for three to five days. Once the cells have recovered, remove the medium and add complete medium containing the appropriate level of eosin to select for stable integration and expression of the TKZO fusion protein.
The eosin concentration can be previously determined as described in the text. Monitor the cells for eosin selection through cell viability. Replacing the medium with fresh eosin containing medium every two to three days.
Once confluent in the six well plate passage pool and expand the cells into larger flasks. A kill curve analysis is performed to establish the minimum lethal dose for the selection reagents used in the study. To avoid undesired phenotypic responses to perform kill curves first plate the cells into each well of a 96 well plate containing 120 microliters of fresh medium the next day.
Add drug to selected wells. Antibiotics G four 18 or pure mycin are used to select for integration of the mirna expression construct or as against acyclovir is used to select against thymidine kinase expression During incubation, examine cell viability for each drug concentration every two days. Replacing the medium containing the selection reagent every three days.
The minimum concentration of selection reagent that causes complete cell death after the desired time should be used for that cell type and experiment after drug concentrations have been determined. Prepare for MI a transfection by growing and expanding the target ID Library cells to attain two times 10 to the seventh cells. Trypsin eyes when cells are at greater than 80%Co fluency.
Transfer the cells to a 15 milliliter sterile screw topped tube and pellet at 200 times G for five minutes. Remove the medium and wash the cell pellet twice as before. Next, for each transfection, add two micrograms of the MIRA expression plasmid to a 0.5 milliliter tube.
Perform NU nuclear affection of the mRNA into the library cells with the same method used for transfection of the target ID library. After the cells have recovered for three to five days, replace the medium with complete medium containing the appropriate level of antibiotic. As determined from the kill curve test.
Monitor the cells for antibiotic selection, replacing the medium with medium containing the antibiotic every two to three days. Once confluent in the six well plate passage pool and expand cells into larger flasks. As before, replace the medium with fresh medium containing the appropriate levels of GCV and antibiotic.
Monitor the cells for thymidine kinase expression and mRNA expression through cell viability and expand the surviving cells in the presence of GCV and antibiotic. Next, prepare genomic DNA from the GCV selected cells. PCR amplify selected library inserts with kit primers and subclone them as described in the written protocol.
Sequencing reactions can then be performed with amplification primers. As a final step, identify gene targets by blast alignment of sequences with the human transcriptome for known transcripts and the human genome for novel transcripts. A mere 3 73 expressing construct was stably transfected into the MC seven library and selected in ganciclovir containing medium to enrich for cells expressing mere 3 73 Targets.
GCV has no effect on MCF seven cells without library because they do not express thymidine kinase. On the other hand, C seven library cells do express thymidine kinase but are not completely killed by ganciclovir at eight or 16 micromolar as shown by the live cells taking up brilliant blue. However, the library cells do stop growing in GCV as evidenced by the color of the phenol red dye in their medium.
The arrested cells do not acidify their medium and the color remains reddish. Instead of changing to orange, yellow target sequences were isolated from the surviving cells. PCR amplified and sequenced.
Nearly 12 million CDNA reads were obtained that mapped to nearly 12, 000 unique genes. Of these unique genes, 234 were considered reliable hits by virtue of hit frequency and were specific to mere 3 73. An initial comparison found that 10 of the unique genes identified with the target ID library are also in the list of previously identified mere 3 73 targets in tar base.
Therefore, these 10 are likely valid targets of mere 3 73 and are currently being characterized to validate selected hits experimentally. After watching this video, you should have a good understanding of how to use the mission target ID library to conduct a high throughput genome wide screen for micro RNA gene targets.
