دورية أكاديمية
أعرض في EDS

Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors.

التفاصيل البيبلوغرافية
العنوان: Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors.
المؤلفون: Moir-Meyer G; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. gemma.moir.meyer@gmail.com., Cheong PL; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. cheong80@gmail.com., Olijnik AA; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. aude-anais.olijnik@ndcls.ox.ac.uk., Brown J; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. jill.brown@imm.ox.ac.uk., Knight S; Wellcome Trust Centre for Human Genetics, Oxford University, Oxford OX3 7BN, UK. sknight@well.ox.ac.uk., King A; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. andrewjohnking@gmail.com., Kurita R; Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, 1-1-3 Shibadaimon, Minato-ku, Tokyo 105-8521, Japan. r-kurita@jrc.or.jp., Nakamura Y; RIKEN BioResource Research Center, Koyadai 3-1-1, Tsukuba 305-0074, Japan. yukio.nakamura@riken.jp., Gibbons RJ; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. richard.gibbons@imm.ox.ac.uk., Higgs DR; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. doug.higgs@imm.ox.ac.uk., Buckle VJ; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. veronica.buckle@imm.ox.ac.uk., Babbs C; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. christian.babbs@imm.ox.ac.uk.
المصدر: Methods and protocols [Methods Protoc] 2018 Jul 30; Vol. 1 (3). Date of Electronic Publication: 2018 Jul 30.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: MDPI AG Country of Publication: Switzerland NLM ID: 101720073 Publication Model: Electronic Cited Medium: Internet ISSN: 2409-9279 (Electronic) Linking ISSN: 24099279 NLM ISO Abbreviation: Methods Protoc Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: Basel, Switzerland : MDPI AG, [2018]-
مستخلص: 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 (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.
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معلومات مُعتمدة: MC_UU_12009/1 United Kingdom MRC_ Medical Research Council; MC_UU_12009 United Kingdom MRC_ Medical Research Council; MC_UU_00016/1 United Kingdom MRC_ Medical Research Council; GN2300 Action Medical Research for Children and the Henry Smith Charity; MC_UU_00016/3 United Kingdom MRC_ Medical Research Council
فهرسة مساهمة: Keywords: CRISPR/Cas9; HUDEP-2 cells; anaemia; homology directed repair
تواريخ الأحداث: Date Created: 20190606 Latest Revision: 20231011
رمز التحديث: 20231011
مُعرف محوري في PubMed: PMC6481050
DOI: 10.3390/mps1030028
PMID: 31164570
قاعدة البيانات: MEDLINE
الوصف
تدمد:2409-9279
DOI:10.3390/mps1030028