Biology subjects

Mikkelsen, M. J.

Publications and source records attributed to Mikkelsen, M. J..

3 recordsLinked to original sources

bioRxiv2024

Analyzing functional heterogeneity of effector cells for enhanced adoptive cell therapy applications

Cellular effector function assays traditionally rely on bulk cell populations that mask complex heterogeneity and rare subpopulations. The Xdrop(R) droplet technology facilitates high-throughput compartmentalization of viable single cells or single-cell pairs in double-emulsion droplets, enabling the study of single cells or cell-cell interactions at an individual level. Effector cell molecule secretion and target cell killing can be evaluated independently or in combination. Compatibility with a wide range of commercial assay reagents allows for single-cell level readouts using common laboratory techniques such as flow cytometry or microscopy. Moreover, individual cells of interest can be viably isolated for further investigation or expansion. Here we demonstrate the application of the double-emulsion droplet technology with a range of cell types commonly utilized for adoptive cell therapy of cancer: peripheral blood mononuclear cells, natural killer cells, tumor-infiltrating lymphocytes, and chimeric antigen receptor T cells. Single-cell compartmentalization offers unparalleled resolution, serving as a valuable tool for advancing the development and understanding of cellular therapy products.

immunology↗

bioRxiv2020

Verification of CRISPR editing by XdropTM Indirect Sequence Capture followed by short- and long- read sequencing

Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the gene editing site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop, a new microfluidic technology that allows targeted enrichment of long regions (~ 100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene edited region in 4 cell lines on long- and short -read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kb large insertions in 3 of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

genomics↗

bioRxiv2018

Xdrop: targeted sequencing of long DNA molecules from low input samples using droplet sorting

Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore such technologies are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of genomic DNA. Xdrop is based on isolation of long DNA fragments in millions of double emulsion (DE) droplets, where the DE droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus (HPV) 18 integrated in the genome of human HeLa cells. The enriched DNA was sequenced both on long-read (PacBio and Oxford Nanopore) and short-read (Illumina) platforms. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex regions of the genome where long-range information is required.

genomics↗