共查询到20条相似文献,搜索用时 31 毫秒
1.
In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI)
to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes
were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI
transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method).
The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression
levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the
ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several
different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained
with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA
concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared
to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared
to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1
(CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression
in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production
of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments,
however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
2.
The successful application of gene therapy depends highly on understanding the properties of gene carriers and their correlation
with the ability to mediate transfection. An important parameter that has been described to improve transfection mediated
by cationic liposomes involves
association of ligands to cationic liposome–DNA complexes (lipoplexes). In this study, ternary complexes composed of 1,2-dioleoyl-3-(trimethylammonium)
propane:cholesterol, plasmid DNA and transferrin (Tf, selected as a paradigm of a ligand) were prepared under various conditions,
namely, in medium with different ionic strengths (HEPES-buffered saline [HBS] or dextrose), at different lipid/DNA (+/–) charge
ratios and using different modes for component addition. We investigated the effect of these formulation parameters on transfection
(in the absence and presence of serum), size of the complexes, degree of DNA protection and extent of their association with
cells (in terms of both lipid and DNA). Our results show that all the tested parameters influenced to some extent the size
of the complexes and their capacity to protect the carried genetic material, as well as the levels of cell association and
transfection. The best transfection profile was observed for ternary complexes (Tf-complexes) prepared in high ionic strength
solution (HBS), at charge ratios close to neutrality and according to the following order of component addition: cationic
liposomes–Tf–DNA. Interestingly, in contrast to what was found for dextrose–Tf-complexes, transfection mediated by HBS-Tf-complexes
in the presence of serum was highly enhanced. 相似文献
3.
Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR
Carapuça E Azzoni AR Prazeres DM Monteiro GA Mergulhão FJ 《Molecular biotechnology》2007,37(2):120-126
A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1
derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires
neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng,
and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases
on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h
post-transfection was around 3 × 104 copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and
around 100 copies/cell were still detected 6 days after transfection. 相似文献
4.
Surfactant-mediated gene transfer for animal cells 总被引:3,自引:0,他引:3
A commercially available cationic surfactant, dimethyl-dioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles.
DDAB easily dissolved in water at 60 °C and formed lipid vesicles at room temperature. The lipid vesicles showed very low
cytotoxicity compared with other cationic surfactants. After the lipid vesicles were mixed with plasmid DNA solution, the
solution was added to mammalian cells. The addition of a nonionic surfactant (Tween 80) to the cationic lipid vesicles at
the weight ratio of 1:1 enhanced transfection efficiency. Adding more or less than the optimal amounts of DNA and lipid vesicles
resulted in decreased transfection efficiency. With the optimal amounts of DNA (pCMVβ) and lipid vesicles, about 90–95% of
CHO-K1 and BHK-21C13 cells transiently expressed β-galactosidase activity 24 h after transfection. By this procedure, stable
transformants around 105 cells corresponding to 10% efficiency could be obtained by one batch transfection.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
5.
An economic method for a rapid estimation of the number of copies of plasmid R6KΔ1 inE. coli cells using lysis of the cells directly on the agarose gel and electrophoretic separation of radioactively labelled plasmid
and chromosomal DNA’s is described. The method, particularly useful for screening purposes, permits detection of both the
CCC and OC forms of plasmids of molar mass 2–150Mg/mol and it can be applied to other bacterial systems. 相似文献
6.
Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both
in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency
of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and
the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett.
10 (2005) 455–470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer.
We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%)
on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer
are highly effective in transfecting SCCVII cells. Approximately 60–70% of SCCVII cells transfected with pCMV.lacZ were positive
for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20–60%) reduced both Metafectene-and GeneJammer-mediated
luciferase expression by ∼30–45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over
90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function
of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory
to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes
is the primary cause of serum inhibition of transfection. 相似文献
7.
Wang B Zhang S Cui S Yang B Zhao Y Chen H Hao X Shen Q Zhou J 《Biotechnology letters》2012,34(1):19-28
Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA
polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy,
gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of
larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes.
The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls
in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control
in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the
nucleus enchanced by chitosan was achieved after 4 h transfection. 相似文献
8.
Yasunori Fukumoto Yuuki Obata Kenichi Ishibashi Naoki Tamura Ikue Kikuchi Kazumasa Aoyama Yasuyuki Hattori Kunihiko Tsuda Yuji Nakayama Naoto Yamaguchi 《Cytotechnology》2010,62(1):73-82
Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although
transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to
experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000)
(PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient
delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and
intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important
for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C,
and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing
the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection
efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately
1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective,
high-efficiency transfection. 相似文献
9.
The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO)
cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture
medium at various times after the initiation of transfection inhibited further cellular uptake of PEI–DNA particles. Using
this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio
of 2:1 (w/w) and a cell density of 2 × 106 cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the
rate and the level of PEI–DNA uptake in serum-free minimal medium were found to be dependent on the PEI–DNA ratio, the cell
density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated
transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian
cells for the purpose of large-scale transient recombinant protein production. 相似文献
10.
Ethyl Pyruvate Inhibits Peroxynitrite-induced DNA Damage and Hydroxyl Radical Generation: Implications for Neuroprotection 总被引:1,自引:0,他引:1
Wei Chen Zhenquan Jia Hong Zhu Kequan Zhou Yunbo Li Hara P. Misra 《Neurochemical research》2010,35(2):336-342
Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism
underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in
the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate
whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical
event leading to peroxynitrite elicited cytotoxicity. Incubation of φX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1),
a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-
dependent manner. The presence of EP (0.5–10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent
fashion. The consumption of oxygen induced by 250 μM SIN-1 was found to be decreased in the presence of EP (0.5–10 mM), indicating
that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite
caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5–10 mM). EPR spectroscopy in combination
with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite,
and that EP at 0.5–10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate
for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation. 相似文献
11.
PEgr–Endostatin–EGFP plasmid was constructed to investigate its expression properties induced by ionizing irradiation and
the effect of pEgr–Endostatin–EGFP gene-radiotherapy on melanoma tumor-bearing mice. The pEgr–Endostatin–EGFP plasmid was
transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of
EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation of three
fractions. Tumor growth was observed for 18 days after treatment. Change of tumor capillary formation was measured with histochemistry
assay at the end of the experiment. The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05–20 Gy.
Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after irradiation with 2 Gy. The
injection of pEgr–Endostatin–EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation
could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The inhibitory effect of pEgr–Endostatin–EGFP
gene-radiotherapy on the growth of B16 melanoma is correlated with the marked decrease of intratumoral vascularization. The
present data point to the potential of an anti-angiogenic approach in gene-radiotherapy of cancer. 相似文献
12.
Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a cytokine used in the treatment of serious conditions resulting
from chemotherapy and bone marrow transplantation such as neutropenia and aplastic anemia. Despite these effects, GM-CSF has
a very short biological half-life, and it requires frequent injection during the treatment. Therefore, the cytokine production
is possible in the body with plasmid-encoded GM-CSF (pGM-CSF) coding for cytokine administered to the body. However, the selection
of the proper delivery system for the plasmid is important. In this study, two different delivery systems, encapsulated plasmid
such as fucoidan–chitosan (fucosphere) and chitosan microspheres, were prepared and the particle physicochemical properties
evaluated. Fucospheres and chitosan microspheres size ranges are 151–401 and 376–681 nm. The zeta potential values of the
microspheres were changed between 8.3–17.1 mV (fucosphere) and +21.9–28.9 mV (chitosan microspheres). The encapsulation capacity
of fucospheres changed between 84.2% and 94.7% depending on the chitosan molecular weight used in the formulation. In vitro plasmid DNA release from both delivery systems exhibited slower profiles of approximately 90–140 days. Integrity of released
samples was checked by agarose gel electrophoresis, and any additional band was not seen. All formulations were analyzed kinetically.
The calculated regression coefficients showed a higher r
2 value with zero-order kinetics. In conclusion, the characterizations of the microspheres can be modulated by changing the
formulation variables, and it can be concluded that fucospheres might be a potential carrier system for the controlled delivery
of GM-CSF encoding plasmid DNA. 相似文献
13.
PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells 总被引:2,自引:0,他引:2
Uegaki K Kanamori Y Kigawa J Kawaguchi W Kaneko R Naniwa J Takahashi M Shimada M Oishi T Itamochi H Terakawa N 《Cell and tissue research》2006,323(3):523-528
PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor.
In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt)
expression, an increase in p27Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling
index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes
in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27,
and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the
proliferation of endometrial carcinoma cells through the induction of contact inhibition. 相似文献
14.
We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection
reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor
scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for
in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium,
optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient
transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing
a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding
bioreactor and the production bioreactor. After 7–8 days the harvest and capture is performed in a one-step operation using
a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final
formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L−1 IgG. 相似文献
15.
Karolina Čepurnienė Paulius Ruzgys Rimantas Treinys Ingrida Šatkauskienė Saulius Šatkauskas 《The Journal of membrane biology》2010,236(1):81-85
DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the
method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved
using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized
in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate
the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer
experiments 50 μl of CHO cell suspension containing 100, 10 or 1 μg/ml of the plasmid were placed between plate electrodes
and subjected to various combinations of HV and LV pulses. The results showed that at 100 μg/ml plasmid concentration LV pulse
delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase
activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 μg/ml) of the plasmid. In
comparison to HV (1,200 V/cm, 100 μs) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold
at 10 μg/ml and fivefold at 1 μg/ml. At 10 μg/ml concentration of plasmid, application of four LV pulses after HV pulse increased
transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer
can be investigated in vitro using HV and LV pulses. 相似文献
16.
Summary Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet
organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm−1) vector DNA concentration (100 μgml−1), carrier DNA concentration (100 μgml−1), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid
vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and
GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when
optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg−1 (protein) min−1. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108.
Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot
analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted
excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S
promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly
except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.
Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the US Department of Agriculture
and does not imply its approval to the exclusion of other products or veudors that may also be suitable. 相似文献
17.
18.
Derouazi M Flaction R Girard P de Jesus M Jordan M Wurm FM 《Biotechnology letters》2006,28(6):373-382
Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of
cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume
was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and
injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding
the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was
cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into
the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection
and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant
cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to
CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell
lines.
Revisions requested 27 October 2005; Revisions received 12 December 2005 相似文献
19.
Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan 总被引:2,自引:0,他引:2
M B Cassidy H Mullineers H Lee J T Trevors 《Journal of industrial microbiology & biotechnology》1997,18(1):43-48
A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient
feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data
processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation
speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual
fed-batch culture. The final cell yield from the automated process reached 60 g L−1 of dry cell weight (OD600 = 120) within 24 h. A plasmid DNA yield of 100 mg L−1 (1.7 mg g−1 cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid
Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process,
acetic acid production was minimal (below 0.01 g L−1) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L−1 was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch
process produced a low cell density averaging 10–12 g L−1 (OD600 = 25–30) and plasmid yields of 5–8 mg L−1 (approximately 0.7 mg g−1 cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result
of a combination of increased cell density, reduced growth rate (μ) from 0.69 h−1 to 0.13 h−1 and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has
proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without
having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales.
Received 22 March 1996/ Accepted in revised form 20 September 1996 相似文献
20.
Norio Gunge Hideki Takata Akira Matsuura Kohsai Fukuda 《Biological procedures online》2003,5(1):29-42
Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats
TG1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary
to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR
(inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences
varied among isolated pTLU-type plasmids, but the TG1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition,
the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends.
This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that
the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids.
Published: February 17, 2003 相似文献