Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. To determine the degradation rate of alphaviral RNA in BHK-21 cells, 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. The findings revealed that the half-life of alphaviral RNA in the cells was approximately 3.5 hours.
Project description:Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused huge economic losses to the duck industry in China since 2010. Moreover, DTMUV can also infect geese, chickens, house sparrows and replicate in in the brain, spleen, liver and kidneys of BALB/c mice, which poses public health concerns. So developing vaccines to combat DTMUV becomes urgent. Baby hamster kidney cell line (BHK-21) is usually employed to produce veterinary vaccine and DTMUV as well as other flaviviruses can be propagated in BHK-21 cells. However, information about mammalian host cell responses to DTMUV infection is limited. In this study, LC–MS/MS coupled to iTRAQ labeling was used to quantitatively identify differentially expressed cellular proteins in DTMUV-infected BHK-21 cells. We identified 192 differentially expressed cellular proteins including 11 upregulated proteins and 8 downregulated at 24 hpostinfection; 25 upregulated proteins and 151 downregulated at 48 h postinfection. Some of these proteins were involved in viral infection.
Project description:We used RNA-sequencing on the Illumina platform to analyze transcriptome profiles of BHK-21 acutely infected with FMDV. In total, we obtained more than 342.8 million raw sequencing reads. And approximately 336.5 million clean reads (98.14%) were acquired by filtering out the adapters and trimming ambiguous reads. We found that cellular response of BHK-21 is related to certain types of alterations in the host cell transcriptome profiles at 24 h post infection.
Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. RNA-seq of 3 different cells was performed to analyze the effect of introducing different versions of RNA replication systems (i.e., repRNA-v3 and repRNA-v4) into BHK-21 cells on endogenous gene expression. Analysis of the transcriptome profiling data indicated that repRNA-v4 stimulated lower interferon signaling and may have lower cytopathicity.