An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

Abstract

Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7–10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1–2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.

The Impact of lncRNAs in Diabetes Mellitus: A Systematic Review and In Silico Analyses

“…The number of lncRNAs differentially expressed between case and control groups from the different included studies varied from 1 ( 23 , 39 , 41 , 43 , 46 – 49 , 52 , 57 , 60 , 64 , 68 , 69 , 73 – 75 , 77 ) to 97,286 ( 58 ), and the sample sizes ranged from 4 ( 66 ) to 370 ( 73 ). Among the 53 studies included in this systematic review, 74% of them analyzed T2DM patients, while 26% did not report which DM type patients had.…”

A CRISPR/Cas9‐based method for targeted DNA methylation enables cancer initiation in B lymphocytes

“…Four days after transfection and puromycin selection, HEK293 and B cells were dissociated and seeded into a single cell/well (96‐well plates) using a limited dilution; cells were diluted to 50 cells in 10 mL, and 100 μL was pipetted into each well of a 96‐well plate 53 . 2 to 3 weeks after seeding, living clones were picked up and genomic DNA was extracted for PCR.…”

Single Cell Metabolomics: A Future Tool to Unmask Cellular Heterogeneity and Virus-Host Interaction in Context of Emerging Viral Diseases

“…Fluorescence-activated cell sorting isolates the target cells specifically labeled with antibody coupled with a fluorescent tags. This technique prefers the samples that are naturally suspended in liquid media, such as blood, bone marrow cells, semen, ovum, secretions, yeast, protoplast, bacteria, and viruses (Ciuffi et al, 2016;Khalil et al, 2017;Janssens et al, 2018;Singh, 2019). Time-lapse fluorescence imaging is a similar type of fluorescence based single cell analysis technique used for monitoring of cell virus interaction (Drayman et al, 2017).…”