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131.
132.
Background
Methods for gene transfer to the cornea that yield high‐level expression without inflammation or trauma are currently lacking. Because electroporation has proven effective for gene transfer in other tissues in terms of expression levels and safety, this study quantitatively evaluated its use in the cornea.Methods
To evaluate the use of electroporation in the mouse cornea, plasmids expressing either luciferase or green fluorescent protein were injected intracorneally or subconjunctivally and square‐wave electric pulses were immediately applied to the eyes. Gene expression was quantified at later times and trauma and inflammation were monitored visually and by measuring interleukin‐6 (IL‐6) production.Results
The application of electric pulses to eyes injected with plasmid resulted in nanogram levels of gene product expression. At an optimal field strength of 200 V/cm, no trauma, corneal edema or inflammation was observed. However, at higher field strengths, corneal damage was detected. Compared with injection of DNA alone, up to 1000‐fold more gene product was produced using electroporation. Expression was detected as early as 6 h post‐electroporation, remained high for 3 days, and decreased by 7 days. Gene expression was detected over the entire surface of the cornea in both epithelial and stromal layers.Conclusions
These results demonstrate that electroporation is an excellent method for delivering genes to multiple cell layers within the mouse cornea and that it results in extremely high levels of gene expression with little, if any, inflammatory response or tissue damage, making this a very useful technique for corneal gene transfer. Copyright © 2001 John Wiley & Sons, Ltd.133.
Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration. 相似文献
134.
Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation. 相似文献
135.
Recombinant transformation vectors (ZPβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter geneβ-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 μF capacitance; 8 ohm resistance and 2 pulses) in the presence
of one of these transformation vectors (100 μg circular DNNml). In either cases 72% of the electroporated eggs successfully
hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosenF
0 individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly
chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of
randomly chosenF
1 progenies was screened for germline transformation. Southern analysis of chosenF
1 progenies revealed the persistence of ZPβypGH or ZpβrtGH in 53% of theF
1 progenies. Southern analyses of chosenF
1 progenies and the frequency (53% ofF
1 ZpβrtGH and 53% ofF
1 ZP{β}ypGH) of transmission revealed the degree of mosaicism inF
0 transformants. Expression was confirmed from the 3–4 times elevated levels of activity of the reporter gene and 30–40% accelerated
growth of transgenicF
0 andF
1 progenies.
Construction of the transgenes was made at the Taiwan National Ocean University, Taiwan and all other work at the Madurai
Kamaraj University, Madurai. 相似文献
136.
137.
Ki Jun Jeong Hyun Sook Lee Sang Yup Lee Yong Keun Chang 《Biotechnology and Bioprocess Engineering》1998,3(1):48-49
A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a
high transformation efficiency. The highest efficiency of 1.6 × 106 transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD600 of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF. 相似文献
138.
R S English J S Lampel TJ Vanden Boom 《Journal of industrial microbiology & biotechnology》1998,21(4-5):219-224
The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electroporation. Various parameters affecting the efficiency of electroporation were examined. The most
critical factor was the extent of spore germination. Electrocompetence was limited to a 4-h period following the initial emergence
of the germ tube. Electroporation efficiencies as high as 2 × 105 CFU μg−1 of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12–14 kV cm−1 with a pulse duration of 15–20 ms. Electrocompetent germlings were stored at −80°C without a significant decrease in transformation
efficiency. The utility of this protocol was demonstrated by isolating a propionyl-CoA carboxylase mutant through targeted
gene disruption and replacement.
Received 3 April 1998/ Accepted in revised form 28 September 1998 相似文献
139.
Søren Kjærulff Dzung Bao Diep Jens Sigurd Okkels Henrik Vibe Scheller John G. Ormerod 《Photosynthesis research》1994,41(1):277-283
Highly efficient and reproducible transformation ofChlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of thepscC gene encoding the cytochromec
551 subunit of the photosynthetic reaction center complex and theaadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without thepscC gene showing that this vector does not replicate inC. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate thepscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate inC. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria. 相似文献
140.
Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.Abbreviations GUS
ß-glucuronidase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- BAP
benzylaminopurine
- PMSF
phenylmethylsulfonyl fluoride
- MES
2[N-Morpholino]ethanesulfonic acid
- HEPES
[N-2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid]
- PAT
Phosphinothricin acetyltransferase
- CTAB
cetyltrimethylammonium bromide 相似文献