Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
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DOI：
10.3390/mps1030028
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发表时间：
2018-09-01
影响因子：
2.4
通讯作者：
Babbs, Christian
中科院分区：
文献类型：
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作者：
Moir-Meyer, Gemma;Cheong, Pak Leng;Babbs, Christian
由人脐静脉衍生的红系前体衍生的永生化红系细胞系（称为HUDEP-2细胞）的产生极大地促进了终末红系发育中细胞过程和基因调控的研究。有效地基因组编辑HUDEP-2细胞和制造克隆系的能力极大地扩展了它们的实用性，因为插入临床相关的突变可以研究可能影响红细胞发育的每种遗传疾病。此外，插入编码短蛋白质标签如Strep、FLAG和Myc的序列允许研究正常和疾病状态下的蛋白质行为。这种方法可用于增强患者细胞的分析，因为可获得大量细胞，并且可以避免对特异性抗体的需要。这种方法可能会导致对疾病机制的深入了解，并提供允许药物发现的试剂。HUDEP-2细胞为现有的永生化红白血病系提供了有利的替代方案，因为它们的核型异常少得多。这些细胞还为广泛的分析提供了足够的材料，因为在培养的9-12天内，可以产生比起始细胞数高4-7倍的体外分化的成红细胞。在这里，我们描述了一种有效的，强大的和可重复的质粒为基础的方法，引入短（
The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4-7 fold higher than starting cell numbers within 9-12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (