Whole-genome multiple displacement amplification from single cells

Spits, Claudia; Caignec, Cédric Le; Rycke, Martine De; Haute, Lindsey Van; Steirteghem, André Van; Liebaers, Inge; Sermon, Karen

doi:10.1038/nprot.2006.326

Cited by 254 publications

(171 citation statements)

References 19 publications

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“…In previous works, glass micropipettes connected to a hydraulic microdrive [37] or Hamilton syringes [36] were used for injection. Here, we used a mouth-controlled pipette system, which enables more accurate and fine manipulations [38], resulting in less leakage throughout the injection. Using this system, we are able to avoid the unexpected incorporation of donor cells into the tissue parenchyma upon penetration of the micropipette.…”

Section: Discussionmentioning

confidence: 99%

Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells

Nagashima

Suzuki

Shitamukai

et al. 2014

Stem Cells and Development

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Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apicobasal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

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Section: Discussionmentioning

confidence: 99%

Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells

Nagashima

Suzuki

Shitamukai

et al. 2014

Stem Cells and Development

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“…hESC colonies were dissociated into single cells using nonenzymatic cell dissociation solution (Sigma-Aldrich). Individual hESCs were manually washed by serial transfer in drops of phosphate-buffered saline (Cell Signaling Technologies) with 0.1% polyvinylpyrrollidone and collected into sterile PCR tubes as previously described 32,33 . The cell's DNA was amplified using the SurePlex DNA Amplification System (BlueGnome, Cambridge, UK), a ligation-mediated PCR-based technique, according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

et al. 2014

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Current knowledge on chromosomal mosaicism in human cell cultures is mostly based on cytogenetic banding methods. The recent development of high-resolution full-genome analysis methods applicable to single cells is providing new insights into genetic and cellular diversity. Here we study the genetic content of 92 individual human cells, including fibroblasts, amniocytes and embryonic stem cells (hESCs), using single-cell array-based comparative genomic hybridization (aCGH). We find that human somatic and embryonic stem cell cultures show significant fractions of cells carrying unique megabase-scale chromosomal abnormalities, forming genetic mosaics that could not have been detected by conventional cytogenetic methods. These findings are confirmed by studying seven clonal hESC sub-lines by aCGH. Furthermore, fluorescent in situ hybridisation reveals an increased instability of the subtelomeric regions in hESC as compared to somatic cells. This genetic heterogeneity may have an impact on experimental results and, in the case of hESC, on their potential clinical use.

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“…Samples for the fecal diversity and E. coli experiments were amplified at LANL using the protocol described in Spits et al (2006). All samples were PCR-amplified using bacterial universal primers (8F/1492R) (Edwards et al 1989;Wilson et al 1990) using AmpliTaq Gold PCR Master Mix (Life Technologies).…”

Section: Mda Pcr and 16s Rdna Analysismentioning

confidence: 99%

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

Fitzsimons

Novotny

et al. 2013

Genome Res.

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The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

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Whole-genome multiple displacement amplification from single cells

Cited by 254 publications

References 19 publications

Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells

Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells

Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

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