In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells

Embleton, M. J.; Gorochov, Guy; Jones, Peter; Winter, Greg

doi:10.1093/nar/20.15.3831

Cited by 133 publications

(57 citation statements)

References 36 publications

(27 reference statements)

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“…SOLiD sequencing (solid.appliedbiosystems.com) is a massively parallel, ligation-mediated sequencing method (Fig. 2) that extends a number of recent developments in nucleic acid chemistry (Kwoh et al 1989;Kwoh and Kwoh 1990;Embleton et al 1992;Macevicz 1998;Tawfik and Griffiths 1998;Ghadessy et al 2001;Dressman et al 2003;Shendure et al 2005;McKernan et al 2006). Tens of millions of 25-50 nt reads are generated by a single run of this sequencing technology.…”

Section: Sequencing and Coveragementioning

confidence: 99%

A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning

Valouev

Ichikawa²,

Tonthat³

et al. 2008

Genome Res.

537

446

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Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.[Supplemental material is available online at www.genome.org. SOLiD raw sequencing data from this study have been submitted to the Short Read Archive at NCBI under accession no. SRA001023.]The regulation of genetic information within eukaryotic cells involves a high degree of specificity both in the availability of individual DNA-binding factors in individual cells, and in availability in the genome of DNA sequences that are their potential targets. Modulating the accessibility of individual DNA sequences are many complex interactions, the most prevalent of which are the interactions between histone octamers and DNA in compacted chromosomes. Each histone core interacts with 147 bp of DNA, which coil 1.7 times around the histone octamer (Luger et al. 1997;Davey et al. 2002) to form the basic unit of chromatin structure, the nucleosome. Since the first description over three decades ago (Kornberg 1974), the nucleosome and its role in gene regulation has been the subject of intensive study and speculation.As we have an increasingly detailed functional view of the genome, the tools of high-throughput molecular characterization have been used to begin obtaining a genome-wide description of nucleosome positions (Satchwell et al. 1986;Yuan et al. 2005;Johnson et al. 2006;Albert et al. 2007;Lee et al. 2007;Peckham et al. 2007;Schones et al. 2008;Shivaswamy et al. 2008). These data have in turn been used in attempts to reveal nucleosome positioning signals in DNA sequence (Satchwell et al. 1986;Ioshikhes et al. 1996;Segal et al. 2006;Yuan and Liu 2008). Although of great interest and value, sequence-based predictions of nucleosome position have been limited to date in their accuracy and resolution.In the litera...

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Section: Sequencing and Coveragementioning

confidence: 99%

A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning

Valouev

Ichikawa²,

Tonthat³

et al. 2008

Genome Res.

537

446

View full text Add to dashboard Cite

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“…To address this issue, we previously applied an in-cell PCR protocol, originally described by Embleton et al (23), to human thyroid-infiltrating CD19 ϩ B cells to obtain in vivo V H /V L gene pairing information (24). In the present study, we report the isolation and characterization of recombinant anti-TPO scFvs generated from this in-cell combinatorial library.…”

mentioning

confidence: 99%

Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

Chapal¹,

Péraldi-Roux²,

Bresson³

et al. 2000

The Journal of Immunology

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In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves’ disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vλ1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves’ disease patients tested were able to strongly inhibit (60–100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.

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“…Previously developed approaches of studying gene expression will not permit further purification of the malignant cells due to (a) cell disruption (Northern blot and colony blot assays), or (b) cell fixation to slides (FISH) (16)(17)(18) The VI, genie segment includes the first and second complementarity deterni-in iing region.s and three franmework regions. The CDRs encode for the regions of the Ig protein which confer antigen binding specificity and display considerable hypervariability compared with the surrounding framework regions which are usually more conserved within each VF, family ( 19).…”

Section: Discussionmentioning

confidence: 99%

Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry.

Cao

Vescio²,

Hong³

et al. 1995

J. Clin. Invest.

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Because it has been difficult to identify and separate malignant cells in human lymphoid malignancies, we have developed a flow cytometry-based fluorescent in situ hybridization (FISH) technique using immunoglobulin (Ig) heavy chain variable region (V H) gene probes. After obtaining the specific V H gene sequence expressed by the multiple myeloma IM-9 cell line and the malignant cells in five multiple myeloma patients, sense and antisense biotinylated single-stranded RNA probes were prepared by transcription from the malignant clone's V H DNA sequences. The cells from the IM-9 cell line and from the mononuclear bone marrow cells of multiple myeloma patients were fixed, hybridized with the above biotinylated RNA probes, incubated with streptavidin-phycoerythrin, and analyzed by FACS' analysis. The myeloma cells stained positive with their own specific antisense V H biotinylated RNA probes, whereas sense and irrelevant antisense biotinylated probes demonstrated only background staining. Dilutional concentrations of the IM-9 cell line with normal bone marrow cells were also accurately quantitated by this procedure. The application of this technique will allow a more accurate assessment of tumor burden in patients with multiple myeloma and should permit an accurate method of tumor cell purification for clinical as well as biological studies. Furthermore, this technological advance should be equally effective at identifying specific V H gene-expressing cells in other lymphoid malignancies, as well as in nonmalignant B cell disorders. (J. Clin. Invest. 1995. 95:964-972.)

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In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells

Cited by 133 publications

References 36 publications

A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning

A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning

Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry.

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