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

Abstract

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...

A designed curved DNA sequence remarkably enhances transgene expression from plasmid DNA in mouse liver

“…We observed a one order of magnitude enhancement in transgene expression by the curved sequences in comparison with ST at 48 h (Figure 2), with much more prominent effects than those induced by the T4 sequence (only 3.5-fold enhancement compared with ST at 48 h). 10 This result agrees with the recent report on the influence of curved sequences in yeast and mammalian cells. 12,24 Thus, the superhelically curved sequence was indicated to be a useful sequence to increase transgene expression in vivo.…”

“…6 The interactions between the plasmid DNA and the histones seem to be one of the factors affecting the intranuclear disposition. 10,11,26,27 Thus, the control of the interactions of the plasmid with histones by the introduction of functional DNA sequences would be important. The left-handedly curved sequence described here is a candidate for such a functional sequence to control nucleosome dynamics on plasmid DNAs and transgene expression in vivo.…”

“…Previously, the short version of the lefthandedly curved sequence (T4) was shown to have the ability to increase transgene expression from plasmid DNA in liver. 10 In the present study, longer versions of the curved sequence (T20--T40) were placed in the upstream region of the tk promoter (Figure 1). We observed a one order of magnitude enhancement in transgene expression by the curved sequences in comparison with ST at 48 h (Figure 2), with much more prominent effects than those induced by the T4 sequence (only 3.5-fold enhancement compared with ST at 48 h).…”

Cytotoxic Effect of Drosophila Deoxynucleoside Kinase Gene on Replicating Plasmid in HeLa Cells

“…This might be due to improved intranuclear disposition of plasmid DNA after replication. Histones could be a key factor for the intranuclear disposition of exogenous DNA 18,19) and replication-dependent loading of histones would enable properly regulated expression from plasmid DNA. In agreement with this speculation, SV40 DNA binds with histones to form minichromosome.…”

Enhanced transgene expression from chromatinized plasmid DNA in mouse liver

“…Thus, the regulation of histone binding to the exogenous DNA would be an important factor for transgene expression, and hence for the intranuclear disposition of the plasmid DNA (Kamiya et al, 2003). Indeed, transgene expression was influenced by the introduction of DNA sequences that modulate histone dynamics into plasmid DNAs (Fukunaga et al, 2012;Kamiya et al, 2007Kamiya et al, , 2009Nishikawa et al, 2003;Sumida et al, 2006). Recently, histones have been used as vehicles for plasmid DNAs (Kaouass et al, 2006 and references therein).…”