کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8480969 | 1551408 | 2018 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Generation of Venus fluorochrome expressing transgenic handmade cloned buffalo embryos using Sleeping Beauty transposon
ترجمه فارسی عنوان
تولید ونوس فلوروکروم بیان جنین های بوفالو شبیه سازی شده بوسیله دست ساز ساخته شده با استفاده از ترانسپسون زیبای خواب
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
علوم کشاورزی و بیولوژیک
علوم کشاورزی و بیولوژیک (عمومی)
چکیده انگلیسی
The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300â¯V, single pulse for 10â¯ms in 2â¯mm cuvette and 1.5-2.0â¯Î¼g transposons with 200-300â¯ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2-3â¯days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400â¯Î¼L of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO2 in air) at 38.5â¯Â°C for 8â¯days and the developmental competence was observed. The percentage of cleaved, 4-8 and 8-16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Tissue and Cell - Volume 51, April 2018, Pages 49-55
Journal: Tissue and Cell - Volume 51, April 2018, Pages 49-55
نویسندگان
Dharmendra Kumar, Papori Sharma, Kennady Vijayalakshmy, Naresh L. Selokar, Pradeep Kumar, Rasika Rajendran, P.S. Yadav,