کد مقاله کد نشریه سال انتشار مقاله انگلیسی ترجمه فارسی نسخه تمام متن
5905286 1159870 2015 7 صفحه PDF سفارش دهید دانلود رایگان
عنوان انگلیسی مقاله ISI
Development of retroviral vectors for insertional mutagenesis in medaka haploid cells
ترجمه فارسی عنوان
توسعه بردارهای رتروویروسی برای جهش درونی در سلول هاپلوئید مدکا
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موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی ژنتیک
چکیده انگلیسی
Insertional mutagenesis (IM) by retrovirus (RV) is a high-throughput approach for interrogating gene functions in model species. Haploid cell provides a unique system for genetic screening by IM and prosperous progress has been achieved in mammal cells. However, little was known in lower vertebrate cells. Here, we report development of retroviral vectors (rvSAchCVgfp, rvSAchCVpf and rvSAchSTpf) and establishment of IM library in medaka haploid cells. Each vector contains a modified gene trapping (GT) cassette, which could extend the mutated cell population including GT insertions not in-frame or in weakly expressed genes. Virus titration determined by flow cytometry showed that rvSAchSTpf possessed the highest supernatant virus titer (1.5 × 105 TU/ml) in medaka haploid cell, while rvSAchCVpf produced the lowest titer (2.8 × 104 TU/ml). However, quantification of proviral DNAs in transduced cells by droplet digital PCR (ddPCR) demonstrated that the “real titer” may be similar among the three vectors. Furthermore, an IM library was established by FACS of haploid cells transduced with rvSAchCVgfp at a MOI of 0.1. A single copy RV integration in the majority of cells was confirmed by ddPCR in the library. Notably, there was a significant decrease of haploid cell percentage after FACS, suggesting potential trapping for survival/growth essential genes. Our results demonstrated successful development of retroviral vectors for IM in medaka haploid cells, serving for haploid genetic screening of host factors for virus infection and genes underlying certain cellular processes in fish model.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Gene - Volume 573, Issue 2, 1 December 2015, Pages 296-302
نویسندگان
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