Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants

Hui, Eric Ka-Wai; Wang, P.-C.; Lo, Szecheng J.

doi:10.1007/s000180050262

Cited by 69 publications

(46 citation statements)

References 57 publications

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“…19 NGF release from NGC0211 cells was more than 10-fold higher than NGC-0295, created using standard liposome transfection techniques (B400 ng 10 À6 cells per 24 h versus B30 ng 10 À6 cells per 24 h).…”

Section: Ngc0211 Cells Secrete High Amounts Of Correctly Processed Ngfmentioning

confidence: 84%

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

et al. 2011

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Nerve growth factor (NGF) is a potential therapeutic agent for Alzheimer's disease (AD) as it has positive effects on the basal forebrain cholinergic neurons whose degeneration correlates with the cognitive decline in AD. We have previously described an encapsulated cell biodelivery device, NsG0202, capable of local delivery of NGF by a genetically modified human cell line, NGC-0295. The NsG0202 devices have shown promising safety and therapeutic results in a small phase 1b clinical study. However, results also show that the NGF dose could advantageously be increased. We have used the sleeping beauty transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD patients, we have validated the bioactivity of devices with NGC0211 and NGC-0295 cells in normal rat striatum as well as in the quinolinic acid striatal lesion model. These preclinical animal studies show that implantation of devices with NGC0211 cells lead to significantly higher NGF output, which in both cases correlate with highly improved potency.

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Section: Ngc0211 Cells Secrete High Amounts Of Correctly Processed Ngfmentioning

confidence: 84%

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

et al. 2011

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“…As discussed in a recent review, a relatively high copy number of integrations is necessary to compensate for the inefficiency of this method. 20 For example, in one successful example of inverse PCR, highly efficient retroviral integration was examined and cells containing integrations were selected using thymidine kinase/HAT selection. 21 Owing to the direct-PCR approach, lack of ligation steps and high sensitivity, repeat-anchored (eg, Alu) PCR is generally preferred over inverse PCR.…”

Section: Discussionmentioning

confidence: 99%

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation

et al. 2004

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Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six-to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay -referred to as repeat-anchored integration capture (RAIC) PCR -that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

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“…in further experiments. Ty1 location: Vectorette PCR (Hui et al 1998), as modified by C. Friddle and D. Botstein (http:/ /www.genomics.princeton. edu/botstein/) was used to determine the chromosomal loca-MATERIALS AND METHODS tion of Ty1his3-AI(96).…”

Section: T He C-value Paradox States That the Haploid Contentmentioning

confidence: 99%

Ty1 Copy Number Dynamics in Saccharomyces

et al. 2005

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To understand long terminal repeat (LTR)-retrotransposon copy number dynamics, Ty1 elements were reintroduced into a "Ty-less" Saccharomyces strain where elements had been lost by LTR-LTR recombination. Repopulated strains exhibited alterations in chromosome size that were associated with Ty1 insertions, but did not become genetically isolated. The rates of element gain and loss under genetic and environmental conditions known to affect Ty1 retrotransposition were determined using genetically tagged reference elements. The results show that Ty1 retrotransposition varies with copy number, temperature, and cell type. In contrast to retrotransposition, Ty1 loss by LTR-LTR recombination was more constant and not markedly influenced by copy number. Endogenous Ty1 cDNA was poorly utilized for recombination when compared with LTR-LTR recombination or ectopic gene conversion. Ty1 elements also appear to be more susceptible to copy number fluctuation in haploid cells. Ty1 gain/loss ratios obtained under different conditions suggest that copy number oscillates over time by altering the rate of retrotransposition, resulting in the diverse copy numbers observed in Saccharomyces.

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Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants

Cited by 69 publications

References 57 publications

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation

Ty1 Copy Number Dynamics in Saccharomyces

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