In the trans kingdom, RNA interference or RNA eye procedure, bacteria are used to deliver short hairpin RNAs or RNAs targeted against the gene of interest MDR one in this protocol into cancer cells, with the aim of silencing the gene of interest by RNAi first esia coli bacteria are transformed with an SH RA expressing vector. Beta one positive cancer cells are then seeded so that the co fluency is 70%to 80%24 hours after seeding. In the next step, the S-H-R-N-A expressing bacteria and the cells are co incubated for two hours.
Finally, the co incubation is terminated by antibiotic treatment of the cancer cells. The results show a downregulation of the gene of interest on the mRNA protein and functional levels through real-time PCR, Western blot analysis, cell proliferation assays, and drug accumulation assays. The main advantage of this technique over other standard procedures like S-I-R-N-A transfection, using oligo effect mean is the effective and the direct delivery of irna into the target cells.
Trans Kingdom RNAi, or TK RNAi technology uses non-pathogenic EIA coli to produce and deliver therapeutic short hairpin RNA or HRNA into target cells to induce RNAi. A first generation TK RNAi mediating vector or trip contains the bacteria phage T seven promoter for expression regulation of a therapeutic SH RNA of interest. Furthermore, trip contains the INV locus from yesinia pseudo tuberculosis that encodes invasive, which permits natural non-invasive bacteria to enter beta one integrin positive mammalian cells.
Tripp also includes the hli, A gene from listeria monocytogenes, which produces listeria lysin O.This enzyme allows the therapeutic HRNA to escape from entry vesicles within the cytoplasm of the target cell. Trop constructs are introduced into a competent non-pathogenic esia coli strain, which encodes T seven RNA polymerase necessary for the T seven promoter driven synthesis of SH RNAs. The bacteria and eukaryotic cells are co incubated, allowing the bacteria to enter the eukaryotic cells.
The SH RNAs are released and into an RNAi pathway. To begin this procedure grow a culture of e coli bacteria transformed with the S-H-R-N-A expressing trip factor with an overnight culture as described in the accompanying written protocol on the day before. Incubating the bacteria with the target cells seed 25 times 10 to the four human gastric carcinoma cells per well in six well dishes.
The number of seeded cells depends on the speed of cell growth of the cell line plan the seeding time so that the co fluency is about 70 to 80%24 hours. After seeding, incubate the seeded cells at 37 degrees Celsius in a 5%carbon dioxide water vapor saturated atmosphere overnight. Before starting the cancer cell infection, measure the optical density of the overnight cultures of the TK RNAi vector containing e coli.
Determine the OD 600, dilute the bacterial solution to an optical density of 0.5 30 minutes before the bacterial co incubation replaced the FCS containing cell culture. Medium of the carcinoma cells with FCS free medium during the 30 minutes period. Wash the bacteria twice with one times PBS by centrifugation at 4, 000 RPM for five minutes at four degrees Celsius and Resus spend in serum free lab vs.
L 15, medium to the original diluted volume. Once the bacteria are resuspended and the cells finish their 30 minutes incubation, add the bacteria to the cancer cells at the desired multiplicity of infection or MRI, which should be in the range of one to 300 to one to 700. Co incubate the bacteria and cancer cells for two hours at 37 degrees Celsius in a 5%carbon dioxide, water vapor saturated atmosphere.
After two hours of co incubation, wash the cancer cells twice with one times PBS and once with serum containing lab bits. L 15 medium supplemented with 100 units per milliliter. Penicillin 100 micrograms per liter streptomycin 2.5 micrograms per milliliter.
Amphotericin 150 micrograms per milliliter gentamicin and 100 micrograms per milliliter. Can mycin continue cultivating the cancer cells at 37 degrees Celsius in a 5%carbon dioxide water vapor saturated atmosphere? The effects of the bacterial treatment can be visualized by fluorescent microscopy, measured by quantitative real-time R-T-P-C-R on the mRNA level, and examined by western blot analysis.
Functional assays like fax analysis and cell proliferation assays can also be used. This representative image was taken three hours after incubating human gastric carcinoma cells with bacteria expressing therapeutic. S-H-R-N-A bacteria can be detected around the nucleus of the treated cell.
In this experiment, downregulation of the expression of the multi-drug resistant gene MDR one was examined by realtime PCR downregulation of mdr. One mRNA of about 70%can be seen after treatment with a therapeutic bacteria. MDR one downregulation also took place on the protein level after bacterial treatment.
MDR one protein levels decreased over time compared with the control cell line. Functional analysis based on the results of the cytotoxicity assay shown here can be used as an indicator for the functioning of the trans kingdom RNAi, while a drug resistant cell line does not respond to the cancer drug. Down aubin, the same cell line infected with bacteria transformed with an anti MDR one S-H-R-N-A expressing plasmid is sensitive to the drug.
Finally, in an anthracycline accumulation assay, the cancer resistant cell line showed little cancer drug accumulation, which was observed in the cancer resistant cells treated with the MDR one SHRA Once mastered. This technique can be applied within 28 hours if it is performed properly following this procedure. Other techniques like R-T-P-C-R can be applied in order to answer several questions like the regulatory effects on the gene of interest.
After watching this video, you should have a good idea about how to apply trans Kingdom RNA interferon by co incubating as HRNA expressing bacteria and better one positive mammalian cells.
