RESUMO
The electrophysiological study is an essential approach to perform the biology and basic medicine research. To achieve the intracellular electrophysiological investigation, electroporation is introduced as an effective and convenient strategy to achieve the intracellular access of electrogenic cells and obtain high-fidelity action potentials. However, seldom platform could provide a quantitative and dynamic strategy to assess the electroporation-induced membrane perforation and recovery during intracellular electrophysiological investigation. Here we develop a high-throughput, sensitive, and stable biosensing platform to assess the evolution of electroporated cell membrane dynamically and quantitatively based on the recorded intracellular electrophysiological signals of cardiomyocytes. Following the electroporation, the extracellular action potentials transiently convert to the intracellular action potentials, whose amplitude rapidly increases to the maximum and then gradually decays. The intracellular action potentials finally convert back to the extracellular action potentials. This biosensing platform can dynamically explore and characterize the evolution procedures of perforation, stabilization, and resealing of the cell membrane by intracellular recordings. Moreover, the effect of electroporation voltages on the cell membrane is segmentally and quantitatively analyzed, demonstrating that a higher electroporation voltage induced a longer resealing time within the safe range of electroporation voltage. We believed that this dynamic and quantitative electroporated membrane evolution biosensing assessment platform will be a promising tool to pave a new avenue to bridge the electrophysiology and electroporated membrane evolution.
Assuntos
Técnicas Biossensoriais , Miócitos Cardíacos , Potenciais de Ação/fisiologia , Fenômenos Eletrofisiológicos , Eletroporação , Miócitos Cardíacos/fisiologiaRESUMO
N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.