Intranuclear disposition of exogenous DNA in vivo: Silencing, methylation and fragmentation

Archives of Biochemistry and Biophysics

Fukunaga

Ohyama

et al. 2007

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The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were . They also showed that histones bound to the left-handedly curved DNA, and that the TATA box was exposed out of the nucleosome core when the curved DNA was located at appropriate positions [9]. This finding prompted us to examine the effects of this left-handedly curved sequence in vivo. In this study, we investigated the in vivo effects of this sequence on plasmids when delivered in a naked form into mouse liver. We found that the position of the left-handedly curved sequence affected the transgene expression by one order of magnitude, without altering the amount of the exogenous DNAs, suggesting the different accessibility of transcriptional factors to the plasmids in vivo. Similar results were observed when plasmids complexed with cationic lipids were delivered into mouse liver. These results indicate that the left-handedly curved sequence on plasmids also determined 5 transgene expression in vivo, and that controlled interactions between the plasmid and the histones are important for the intranuclear disposition. Materials and Methods MaterialsOligodeoxyribonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) in purified forms. The pLHC4/TLN-6, pLHC4/TLN-16, pLHC4/TLN+47, and pST0/TLN-7 plasmids, containing the thymidine kinase (tk) promoter and the luciferase gene (Fig. 1) [9], were amplified in Escherichia coli strain DH5α and purified with a Qiagen (Hilden, Germany) EndoFree Plasmid Mega kit. Hydrodynamics-based injectionPlasmid DNA (20 µg in 2 ml of saline) was injected into the tail vein of male six week-old Balb/c mice within 5 sec [10,11]. The livers were harvested from the injected mice at various time points, and the luciferase activity and the amount of the exogenous DNA were measured, as described below. Luciferase activity 6Livers were minced with scissors and homogenized completely in Lysis Buffer (100 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.8). After centrifugation at 13,000 g for 10 min at 4°C, the supernatant was examined for luciferase activity, using a Luciferase Assay Systems kit (Promega, Madison, Wisconsin, USA). Isolation of nuclear DNA and Quantitative PCRLivers were homogenized in phosphate-buffered saline (PBS).After centrifugation at 2,500 g for 5 min at 4°C, the pellet was washed three times with PBS. The pellet was resuspended in DNA Lysis Buffer (100 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.5% (w/v) IGEPAL-CA630, pH 7.4) [12]. After centrifugation at 1,400 g for 5 min at 4°C, the pellet was washed three times with DNA Lysis Buffer. The intranuclear DNA was extracted with the SepaGene reagent (Sanko Jun-yaku, Tokyo, Japan).Quantitative PCR (Q-PCR) was performed using an ABI 7500 real time PCR system, and SYBR-Green...

Section: (Introduction)mentioning

confidence: 99%

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

Archives of Biochemistry and Biophysics

Fukunaga

Ohyama

et al. 2007

Self Cite

“…In the case of naked plasmid DNA delivered by the hydrodynamic tail vein injection, most of the delivered DNA was degraded within two days (Ochiai et al, 2006). Thus, TD fragment delivered in the naked form would also be fragmented within two days.…”

Section: Discussionmentioning

confidence: 99%

Targeted sequence alteration of a chromosomal locus in mouse liver

International Journal of Pharmaceutics

Uchiyama

Piao

et al. 2010

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Targeted sequence alteration would be an attractive method in gene therapy and biotechnology. To achieve in vivo targeted sequence alteration, a tailed duplex DNA consisting of annealed 35mer and 794mer single-stranded DNAs was delivered by means of hydrodynamic tail vein injection into liver of transgenic mouse harboring a reporter gene (the rpsL gene) in its genome. The tailed DNA was designed for a conversion of ATC to AGC at codon 80 of the rpsL transgene. The anticipated TG sequence alteration was induced in the transgene in the liver with an efficiency of ~0.1%.These results demonstrate the significant potential of this method for applications in gene therapy and biotechnology.

“…The pYK-CMV-luc plasmid DNA (Ochiai et al, 2006) was amplified in Escherichia coli strain DH5α, and was purified with a Qiagen (Hilden, Germany) EndoFree Plasmid Mega kit.…”

Section: Methodsmentioning

confidence: 99%

“…Quantitative PCR (Q-PCR) was performed using an ABI 7500 real time PCR system (Applied Biosystems, Foster City, California, USA) and SYBR-Green chemistry, as described previously (Ochiai et al, 2006), using the following primers:…”

Section: Isolation Of Nuclear Dna and Quantitative Pcrmentioning

confidence: 99%

Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3

International Journal of Pharmaceutics

Goto

Kanda

et al. 2010

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The intranuclear disposition of plasmid DNA is extremely important for transgene expression. Exogenous histones have been used as carriers of plasmid DNA in histone-mediated gene delivery. In this study, the effects of exogenous histone H3 complexed with plasmid DNA on transgene expression efficiency were examined. The plasmid−histone complexes in various ratios were transfected into HeLa cells by osmotic pressure.Histone H3 suppressed transgene expression in the nucleus in a dose-dependent manner. Our results suggest that the histone-mediated gene delivery is unlikely to be useful, from the viewpoint of the intranuclear disposition.