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首页> 外文期刊>BMC Biotechnology >Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0
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Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

机译：大鼠杂交瘤细胞YB2 / 0中表达的α1,6-岩藻糖基转移酶基因（fut8）的分子克隆，表征，基因组组织和启动子分析

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Background The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene. Results The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells. Conclusion Altogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.
机译：背景大鼠杂交瘤细胞系YB2 / 0似乎是大规模生产低岩藻糖重组mAb的良好候选者，因为它的fut8基因表达低于其他常用啮齿动物细胞系。然而，在生产培养条件下观察到重组mAb的岩藻糖含量的重要变化。为了提高我们对YB2 / 0岩藻糖基化能力的认识，我们已经克隆并鉴定了大鼠fut8基因。结果采用聚合酶链反应和5'RNA-Ligase介导的RACE方法从YB2 / 0细胞中克隆了编码大鼠α1,6-岩藻糖基转移酶（FucT VIII）的cDNA。 cDNA包含1728 bp的开放阅读框，编码575个氨基酸序列，分别与人和猪的直系同源物具有94％和88％的同一性。在COS-7细胞中表达的重组蛋白对人去唾液酸-agalacto-载脂蛋白转铁蛋白表现出α1,6-岩藻糖基转移酶活性。大鼠fut8基因位于6 q染色体上，跨度超过140 kbp。它包含9个编码外显子和4个5'-非翻译外显子。 FISH分析显示YB2 / 0核中fut8的异质拷贝数，平均拷贝数为2.8±1.4。 YB2 / 0 fut8基因表达为两个主要的转录本，通过使用不同的启动子和选择性剪接，它们在第一个未翻译的外显子中有所不同。萤光素酶测定允许定义控制两个转录本起始的最小促进区域，如双链Taqman QPCR分析所示，它们在YB2 / 0中差异表达。对分别控制T1和T2转录本转录的上游外显子E-2和E-3最小启动子区域的生物信息学分析证明了潜在的转录阻遏物的几个共有序列。用转录因子表达载体对Rat2细胞进行瞬时转染，可将KLF15鉴定为Rat2细胞中T1转录的推定阻遏物。结论总之，这些数据有助于更好地了解YB2 / 0中fut8的表达，这将有助于更好地控制这些细胞中产生的重组mAb的岩藻糖基化。

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