Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies.

In an era of cost-efficient gene synthesis and high-throughput construct assembly, the onus of scientific experimentation is on the rate of in vivo testing for the identification of top performing candidates or designs. Assay platforms that are relevant to the species of interest and in the tissue of choice are highly desirable. A protoplast isolation and transfection method that is compatible with a large repertoire of species and tissues would be the platform of choice. A necessary aspect of this high-throughput screening approach is the need to handle many delicate protoplast samples at the same time, which is a bottleneck for manual operation. Such bottlenecks can be mitigated with the use of automated liquid handlers for the execution of protoplast transfection steps. The method described within this chapter utilizes a 96-well head for simultaneous, high-throughput initiation of transfection. While initially developed and optimized for use with etiolated maize leaf protoplasts, the automated protocol has also been demonstrated to be compatible with other established protoplast systems, such as soybean immature embryo derived protoplast, similarly described within. This chapter also includes instructions for a sample randomization design to reduce the impact of edge effects, which might be present when microplates are used for fluorescence readout following transfection. We also describe a streamlined, expedient, and cost-effective protocol for determining gene editing efficiencies using the T7E1 endonuclease cleavage assay with a publicly available image analysis tool.

How Reliable are Test Numbers for Revealing the COVID-19 Ground Truth and Applying Interventions?

The number of confirmed cases of COVID-19 is often used as a proxy for the actual number of ground truth COVID-19-infected cases in both public discourse and policy making. However, the number of confirmed cases depends on the testing policy, and it is important to understand how the number of positive cases obtained using different testing policies reveals the unknown ground truth. We develop an agent-based simulation framework in Python that can simulate various testing policies as well as interventions such as lockdown based on them. The interaction between the agents can take into account various communities and mobility patterns. A distinguishing feature of our framework is the presence of another 'flu'-like illness with symptoms similar to COVID-19, that allows us to model the noise in selecting the pool of patients to be tested. We instantiate our model for the city of Bengaluru in India, using census data to distribute agents geographically, and traffic flow mobility data to model long-distance interactions and mixing. We use the simulation framework to compare the performance of three testing policies: Random Symptomatic Testing (RST), Contact Tracing (CT), and a new Location-Based Testing policy (LBT). We observe that if a sufficient fraction of symptomatic patients come out for testing, then RST can capture the ground truth quite closely even with very few daily tests. However, CT consistently captures more positive cases. Interestingly, our new LBT, which is operationally less intensive than CT, gives performance that is comparable with CT. In another direction, we compare the efficacy of these three testing policies in enabling lockdown, and observe that CT flattens the ground truth curve maximally, followed closely by LBT, and significantly better than RST.