Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences

Lusky, Monika; Botchan, Michael R.

doi:10.1038/293079a0

Cited by 655 publications

(328 citation statements)

References 16 publications

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“…However, it should be noted that good expression can be detected at 24 hours with as few as 10 copies of pGFPΔSV40 injected into the cytoplasm, a finding that suggests that very little degradation of the plasmid occurs. This conclusion is supported by others who have found little degradation of plasmid DNA in microinjected or transfected cells (15,34), although there is no consensus in the field regarding DNA degradation in transfections (35). An alternative explanation for our finding that the hCMViep and RSV LTR are incapable of targeting plasmids to the nucleus is that these plasmids are indeed capable of mediating nuclear import of plasmid DNA, but do so very slowly, requiring greater than 24 hours.…”

Section: Discussionsupporting

confidence: 68%

Sequence Requirements for Plasmid Nuclear Import

Dean

Müller³

et al. 1999

Experimental Cell Research

215

244

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SUMMARYThe nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of non-viral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366 bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72 bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in non-viral vectors may greatly increase their ability to be transported into the nucleus, especially in non-dividing cells.

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Section: Discussionsupporting

confidence: 68%

Sequence Requirements for Plasmid Nuclear Import

Dean

Müller³

et al. 1999

Experimental Cell Research

215

244

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“…Plasmid transfer: The transfer vector is based on plasmid pML2, 67 which was derived from pBR322. A EcoRI linker was inserted into pML2 leading to plasmid pTG1E which therefore contains a single EcoRI site, an E. coli origin of replication and the ␤-lactamase gene conferring ampicillin resistance.…”

Section: Elispot (T Cell Activity)mentioning

confidence: 99%

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice

et al. 2000

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Intramuscular administration of plasmid expressing fulllength human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejec-

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“…These elements were cloned in EcoRIlBamHI digested pML [9]. A human ubiquitin gene (Ub C) promoter [ 101 was cloned at the 5 ' -end of the expression unit.…”

Section: Dna Consinictionsmentioning

confidence: 99%

Partial processing of the neuropeptide Y precursor in transfected CHO cells

et al. 1990

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The activation of regulatory peptides by post-translational modification of their biosynthetic precursors is generally thought to occur only in neuroendocrine cells. We have selected clones of Chinese hamster ovary cells, a non-neuroendocrine cell line, which were transfected with a eukatyotic expression vector coding for the precursor for neuropeptide Y. Although the majority of the immunoreactive NPY was found in the form of pro-NPY, some degree of intracellular proteolytic processing of the precursor occurred in all clones. Part of the intracellular NPY immunoreactivity was even correctly amidated. Extracellular degradation of pro-NPY in the tissue culture medium generated i~uno~acti~ty which corresponded in size to NPY. It is concluded that precursor processing can occur in non-neur~nd~~ne cells both as a biological process within the cells and as apparent processing, degradation in the tissue culture. medium.

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Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences

Cited by 655 publications

References 16 publications

Sequence Requirements for Plasmid Nuclear Import

Sequence Requirements for Plasmid Nuclear Import

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice

Partial processing of the neuropeptide Y precursor in transfected CHO cells

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