Factor(s) required by EBV transformed lymphocytes to grow under limiting dilution conditions

Negri, C.; Chiesa, Roberto; Ricotti, G.

doi:10.1007/bf00365928

Cited by 5 publications

(3 citation statements)

References 19 publications

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“…These observations are consistent with (Fig. 6A to C) (29,35). Conventional inoculation of TMC resulted in a transformation efficiency of around 60% when starting with 200 cells per well.…”

Section: Spinoculation With Concentrated Virus Substantially Increasesupporting

confidence: 90%

“…The wild-type B95.8 EBV was used to exclude potential interference of the recombinant EBV with the transformation process. To provide similar culture conditions for purified B cells and mononuclear cells, we added B-cell-depleted TMC as a feeder layer for ex vivo EBV-infected naïve and memory B cells immediately after spinoculation (29). One half of the medium was changed every 3 days for 4 weeks in all wells initially used.…”

Section: Methodsmentioning

confidence: 99%

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Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin

Dorner

Zucol

Berger

et al. 2008

J Virol

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“…These observations are consistent with (Fig. 6A to C) (29,35). Conventional inoculation of TMC resulted in a transformation efficiency of around 60% when starting with 200 cells per well.…”

Section: Spinoculation With Concentrated Virus Substantially Increasesupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin

Dorner

Zucol

Berger

et al. 2008

J Virol

View full text Add to dashboard Cite

show abstract

“…Cultured Human Lymphocytes-Lymphocytes were isolated from the heparinized blood of a patient homozygous for the ⑀2 allele and immortalized with Epstein-Barr virus (20). Transformed cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Sigma) and 2 mM L-glutamine.…”

Section: Recombinant Cho Cells-chomentioning

confidence: 99%

Gene Correction of the Apolipoprotein (Apo) E2 Phenotype to Wild-type ApoE3 by in Situ Chimeraplasty

Tagalakis

Graham

Riddell

et al. 2001

Journal of Biological Chemistry

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Apolipoprotein (apo) E is a polymorphic plasma protein, synthesized mainly by liver. Here, we evaluate whether synthetic DNA-RNA oligonucleotides (chimeraplasts) can convert a dysfunctional isoform, apoE2 (C 3 T, R158C), which causes Type III hyperlipidemia and premature atherosclerosis, into apoE3. First, we treated recombinant Chinese hamster ovary cells stably secreting apoE2 with a 68-mer apoE2 to apoE3 chimeraplast. About one-third of apoE2 was converted to apoE3, and the repair was stable through 12 passages. Subcloning treated cells produced both apoE2 and apoE3 clones. Direct sequencing and reverse transcription polymerase chain reaction confirmed the genotype, whereas phenotypic change was verified by isoelectric focusing and immunoblotting of secreted proteins. Second, we established that the APOE2 gene can be targeted both in vivo, using transgenic mice overexpressing human apoE2, and in chromosomal context, using cultured lymphocytes from a patient homozygous for the ⑀2 allele. We conclude that chimeraplasty has the potential to convert the apoE2 mutation in patients with Type III hyperlipidemia to apoE3. Apolipoprotein (apo)1 E is a 34-kDa polymorphic protein that has anti-atherogenic actions by clearing remnant lipoproteins and promoting cholesterol efflux from cells (1-4). Low apoE is a risk factor for coronary artery disease (5), and apoE deficiency results in severe hyperlipidemia and atherosclerosis (6, 7). By contrast, infusing apoE into hyperlipidemic rabbits regresses atherosclerotic lesions (8), and apoE transgenic mice resist diabetic or dietary hyperlipidemia (9, 10). The three common isoforms of apoE differ in two amino acid positions; apoE2 (Cys-112 and Cys-158), apoE3 (Cys-112 and Arg-158), the most prevalent or wild-type protein, and apoE4 (Arg-112 and Arg-158). The rarest variant, apoE2, is the primary molecular cause of Type III hyperlipidemia (11), which is characterized by accumulation of remnant lipoproteins and premature heart disease.Approaches to treat or prevent atherosclerosis include systemic gene therapy, using viral or non-viral delivery strategies, to overexpress proteins that inhibit atherogenesis or stabilize lesions. Indeed, adenoviral-mediated apoE gene transfer ameliorates experimental hyperlipidemia and atherosclerosis (12, 13). The recent development of targeted gene repair, using synthetic DNA-RNA oligonucleotides (chimeraplasts), offers another possibility (14 -16). Chimeraplasts contain short regions of correcting DNA bounded by long stretches of 2Ј-Omethyl RNA; hairpin loops and GC clamps are also incorporated to make the structure self-associating. These features promote strong, specific binding to the target genomic sequence and allow the DNA repair machinery of the cell to identify and correct the point mutation in situ (15).Here, we apply chimeraplasty to recombinant Chinese hamster ovary (CHO) cells secreting human apoE2 and show that the gene conversion of apoE2 to apoE3 is stable and permanent at the DNA, mRNA, and protein level. In addition, we demonst...

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Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

Farrell

Kalogerakis

Behie

1994

Cell Culture Engineering IV

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Factor(s) required by EBV transformed lymphocytes to grow under limiting dilution conditions

Cited by 5 publications

References 19 publications

Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin

Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin

Gene Correction of the Apolipoprotein (Apo) E2 Phenotype to Wild-type ApoE3 by in Situ Chimeraplasty

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

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