Proof of Concept

Our previous experimental results were the same as we expected and proved our experimental assumptions. Our plan is to extend the diagnostic tools to community hospitals or examination centers closer to patients in remote areas. This avoids the inconvenience of testing the results in larger hospitals and reduces the waiting time for the result. In order to apply our project in the real world, we used cell free system to verify the splicing sensor.

1. Verifying ZNF91-LUC Sensor in a cell-free system.

After the intracellular transfection test, we used a cell-free assay to test the effect of these plasmid sensors in vitro. It was mentioned earlier that the ZNF91-LUC plasmid sensor is more sensitive. So we designed exon-skipping LUC reporters which contains parts of the ZNF91 intronic and exonic sequences in the luciferase gene in the MCS region of pT7CFE1 expression vector. Positive clones were screened by solid plates containing ampicillin and sequencing. Plasmids were expressed in vitro using the 1-step Coupled Human IVT Kit for DNA (Thermo Fisher). The kit is a unique protein expression system based on Hela cell lysates for in vitro translation. Adding the plasmids to a mixture of HeLa cell lysate, accessory proteins, reaction mix, then, 293T cell lysates were added to simulate mRNA splicing. This system can be incubated at 30℃ for 90 minutes for protein. The proteins translated in vitro were assayed for luciferase activity as described previously (Table 1, Fig 1). We can see from the results that ZNF91-LUC Sensor can work in a cell-free system. These results imply that we can use the splicing sensor without transfection, which brings great convenience for future clinical application.


Table 1. The value of fluorescence of pT7CFE1-ZNF91-LUC protein expressed in vitro

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Fig 1. The relative luciferase activity of pT7CFE1-ZNF91-LUC protein expressed in vitro.

2. Dry lab design

We found that Adeno-Associated Virus Type 2(AAV Type-2)can infect primary bone marrow cells. Hence, we assume that combine splicing reporters (MAP3K7-LUC or ZNF91-LUC) and a plasmid to construct a recombinant AAV virus. After collecting the virus supernatant and concentrating the virus, the virus was directly used to infect primary bone marrow cells. We collect infected primary bone marrow cells, to lysis and collect protein samples for luciferase or fluorescence tests. Thus, we can directly and conveniently get the result, and therefore help to diagnosis MDS.

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Fig. 2. RNA splicing reporter using AAV infection.

Firstly, we transfect the MAP3K7-LUC /ZNF91-LUC and two packaged plasmids into the cell of 293T, then collect AAV-type2 virus supernatant samples 48 hours later. Secondly, we extract samples from patients' bone marrow cells, and use virus supernatant samples to infect bone marrow cells. Thirdly, after 48-72 hours infection, we start to lysis the cells and collect proteins. Finally, we add substrate buffer, detect the fluorescence values and get the corresponding sample results.