An efficient method for routine epstein-barr virus immortalization of human B lymphocytes

Wall, Florence E.; Henkel, Richard D.; Stern, Michael P.; Jenson, Hal B.; Moyer, Mary Pat

doi:10.1007/bf02633976

Cited by 35 publications

(22 citation statements)

References 32 publications

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“…Cultures then were divided into 150-l aliquots in 96-well plates, 50 l of B95-8 conditioned media [containing Epstein-Barr virus (EBV) and generated from B95-8 cells, as described (15), except that IMDM with 20% (vol͞vol) FBS was used] was added to each well, and cytokines were replenished. When media began to acidify, volumes were doubled by the addition of lymphoid growth medium, and cultures were transferred into progressively larger wells.…”

Section: Methodsmentioning

confidence: 99%

Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors

Josephson

Vassilopoulos

Trobridge

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The efficiency of gene transfer into human hematopoietic stem cells by oncoretroviral vectors is too low for effective gene therapy of most hematologic diseases. Retroviral vectors based on the nonpathogenic foamy viruses (FV) are an alternative gene-transfer system. In this study, human umbilical cord blood CD34 ؉ cells were transduced with FV vectors by a single 10-h exposure to vector stocks and then injected into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD͞SCID) mice. At 5-7 weeks after transplantation, high transgene expression rates were observed in engrafted human hematopoietic cells, including over 60% of clonogenic progenitors. Significant transgene silencing did not occur. We developed an approach for expanding human cell populations derived from transplanted mice to show that multiple SCID repopulating cells (SRCs) had been transduced, including some that were capable of both lymphoid and myeloid differentiation. These findings demonstrate for the first time that human pluripotent (lympho-myeloid) hematopoietic stem cells repopulate NOD͞SCID mice and can be efficiently transduced by FV vectors. S pumaviruses or foamy viruses (FV) are nonpathogenic retroviruses with a wide tissue tropism commonly found in mammals (1, 2), although humans are not a natural reservoir of infection (3, 4), and there is no evidence of human-to-human transfer (5, 6). FV vectors transduce nondividing cells more efficiently than oncoretroviral vectors, are not inactivated by human serum, and have a large packaging capacity (7). We previously developed methods for the production of high titer, helper-free FV vectors (8) and found that they could efficiently transduce murine hematopoietic stem cells (HSCs) (9). However, the high HSC transduction rates obtained in mice by other vector types have not been reproduced in primates, prompting us to test FV vectors in human cells.One model currently used to study human hematopoiesis is xenotransplantation of immunodeficient nonobese diabeticsevere combined immunodeficiency (NOD͞SCID) mice (10, 11). These mice support multilineage human hematopoiesis for several weeks, and the low transduction rates of SCID repopulating cells (SRCs) by oncoretroviral vectors indicate that it is an excellent preclinical gene therapy model of human repopulating cells (12). SRCs have proliferative capacities, repopulation kinetics, and cell-surface phenotypes that suggest they are true HSCs (12-14); however, multipotential differentiation has not been demonstrated conclusively by analysis of vector integration patterns in lineage-specific progeny cells. Here, we show that FV vectors efficiently transduce SRCs, and that clonal lymphomyeloid repopulation occurs with marked cells as expected for transduced pluripotent HSCs. Materials and MethodsHematopoietic Cell Culture and Transductions. Human CD34 ϩ cells were isolated from umbilical cord blood under an Institutional Review Board-approved protocol by using a CD34 ϩ cell isolation kit as per the manufacturer's instructions ...

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Section: Methodsmentioning

confidence: 99%

Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors

Josephson

Vassilopoulos

Trobridge

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…22 EBVtransformed lymphoblastoid cell lines (EBV-LCLs) from patients and controls were grown at 37 1C and 7.5% CO 2 in RPMI 1640 medium (Gibco, Breda, The Netherlands) containing 10% (v/v) fetal calf serum (Sigma, Zwijndrecht, The Netherlands), 1% 10 U/ml penicillin and 10mg/ml streptomycin (Gibco), and 1% GlutaMAX (Gibco). Twenty-four hours before emetin treatment, cells are centrifuged at 200 Â g for 5 min at room temperature and resuspended in fresh medium to a density of 0.7 million cells per ml.…”

Section: Cell Culturingmentioning

confidence: 99%

A 3-base pair deletion, c.9711_9713del, in DMD results in intellectual disability without muscular dystrophy

Brouwer¹,

Nabuurs

Verhaart

et al. 2013

Eur J Hum Genet

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We have identified a deletion of 3 base pairs in the dystrophin gene (DMD), c.9711_9713del, in a family with nonspecific X-linked intellectual disability (ID) by sequencing of the exons of 86 known X-linked ID genes. This in-frame deletion results in the deletion of a single-amino-acid residue, Leu3238, in the brain-specific isoform Dp71 of dystrophin. Linkage analysis supported causality as the mutation was present in the 7.6 cM linkage interval on Xp22.11-Xp21.1 with a maximum positive LOD score of 2.41 (MRX85 locus). Molecular modeling predicts that the p.(Leu3238del) deletion results in the destabilization of the C-terminal domain of dystrophin and hence reduces the ability to interact with b-dystroglycan. Correspondingly, Dp71 protein levels in lymphoblastoid cells from the index patient are 6.7-fold lower than those in control cell lines (P ¼ 0.08). Subsequent determination of the creatine kinase levels in blood of the index patient showed a mild but significant elevation in serum creatine kinase, which is in line with impaired dystrophin function. In conclusion, we have identified the first DMD mutation in Dp71 that results in ID without muscular dystrophy.

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“…"Immortalization" of peripheral blood B lymphocytes with EpsteinBarr virus utilized method 2 of Wall et al (1995); media, passaging and cryopreservation were as described by these authors except that lots of heat-inactivated (56°C for 30 min) fetal bovine serum (FBS), used at 16% concentration, were pretested for ability to support growth of established lymphoblastoid cell lines at concentrations of approximately 200 000 cells per ml. Cryopreservation (freezing at approx.…”

Section: Introductionmentioning

confidence: 99%

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants

et al. 1997

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Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

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An efficient method for routine epstein-barr virus immortalization of human B lymphocytes

Cited by 35 publications

References 32 publications

Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors

Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors

A 3-base pair deletion, c.9711_9713del, in DMD results in intellectual disability without muscular dystrophy

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants

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