Saturday, February 18, 2017
Exhibit Hall (Hynes Convention Center)
Mohamad Najia, Harvard-MIT Health Sciences & Technology, Cambridge, MA
Background: Cellular development, morphology and function are governed by the precise patterning of gene expression. Transcriptome-wide profiling of cell populations has enabled rich biological insights that characterize the molecular state of cells. Recently, single cell RNA-sequencing has furthered our understanding of the contributions of individual cells to mammalian biology. Although informative, these measurements are inherently static snapshots of dynamic cellular processes. Transitions in cell state and function over time are crucial to cellular biology, and enabling comprehensive investigation of the temporal dynamics within the transcriptome is of intense interest. However, tools for non-destructive in situ analysis of the transcriptome of single cells are currently lacking. Thus, the objective of this study is to engineer cellular technologies to non-destructively analyze the transcriptome of single cells over time. Results:We have engineered cells to “self-report” representative samples of their transcriptome through the packaging and extracellular export of RNAs within virus-like particles (VLPs). Gag viral proteins were fused to an MS2 binding domain and a library of cell-specific barcodes with an MS2 hairpin loop were lentivirally transduced into the genome of single cells. Upon transcription, the cell barcode sequence hybridizes to the poly(A) tail of mRNAs through a uridylated hybridization sequence and is packaged into Gag-MS2 VLPs through the binding of the MS2 RNA hairpin to the MS2 binding domain. Purification of VLPs from the cellular media and RNA-sequencing reveals single-cell resolved, temporal transcriptomic profiles. RNase treatment of purified VLPs and subsequent RT-qPCR amplification of GAPDH mRNA demonstrated RNA stability within VLPs. Methods:Gag-MS2 fusion protein and cell-barcode libraries were generated via Gibson Assembly. Stable genomic integration of the cell-barcode libraries was achieved through lentiviral vectors and Gag-MS2 constructs were transiently expressed through Lipofectamine-based transfection of HEK293T cells. NGS libraries were prepared via the Smart-seq2 protocol and sequenced on an Illumina NextSeq. Conclusions: The preliminary results from this study demonstrate that engineered VLPs can temporally export mammalian mRNA and that packaged mRNA is stable within VLPs. Cell-specific barcodes expressed as RNAs enable single-cell resolved temporal transcriptomic analysis. This technology has strong potential to enable dissection of transcriptomic trajectories during cell-state transitions and differentiation