Abstract
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.
Highlights
Studying disease pathogenesis affecting terminal erythroid differentiation requires model cellular systems capable of generating sufficient material to allow genomic, transcriptomic, proteomic and cell biology approaches
DNA can be extracted from the cell pellets in microcentrifuge tubes and screened by polymerase chain reaction (PCR) amplification and Sanger sequencing coupled with TIDER software [24] or restriction digest to determine correct integration of the required genomic modification
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Summary
Studying disease pathogenesis affecting terminal erythroid differentiation requires model cellular systems capable of generating sufficient material to allow genomic, transcriptomic, proteomic and cell biology approaches. The use of patient cells often provides insufficient material for many approaches To deal with these problems, a number of cell lines have been used to study terminal erythroid differentiation in the laboratory. These include the human erythroleukaemia cell lines termed K562 and HEL [1,2] and the mouse equivalent, termed MEL [3] Study of these cell lines in conjunction with small interfering RNA (siRNA) mediated knockdown of specific genes has provided insight into normal and aberrant terminal erythroid differentiation, these lines are extremely aneuploid and harbour chromosome rearrangements that hinder genomic manipulation and may adversely affect terminal differentiation [4]. Here we report a robust and efficient protocol delivering Cas via plasmids and the template for homology
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