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|Title:||Robust imaging and gene delivery to study human lymphoblastoid cell lines|
|Citation:||Journal of Human Genetics, 2018; 63(9):945-955|
|Publisher:||Nature Publishing Group|
|Lachlan A. Jolly, Ying Sun, Renée Carroll, Claire C. Homan, Jozef Gecz|
|Abstract:||Lymphoblastoid cell lines (LCLs) have been by far the most prevalent cell type used to study the genetics underlying normal and disease-relevant human phenotypic variation, across personal to epidemiological scales. In contrast, only few studies have explored the use of LCLs in functional genomics and mechanistic studies. Two major reasons are technical, as (1) interrogating the sub-cellular spatial information of LCLs is challenged by their non-adherent nature, and (2) LCLs are refractory to gene transfection. Methodological details relating to techniques that overcome these limitations are scarce, largely inadequate (without additional knowledge and expertise), and optimisation has never been described. Here we compare, optimise, and convey such methods in-depth. We provide a robust method to adhere LCLs to coverslips, which maintained cellular integrity, morphology, and permitted visualisation of sub-cellular structures and protein localisation. Next, we developed the use of lentiviral-based gene delivery to LCLs. Through empirical and combinatorial testing of multiple transduction conditions, we improved transduction efficiency from 3% up to 48%. Furthermore, we established strategies to purify transduced cells, to achieve sustainable cultures containing >85% transduced cells. Collectively, our methodologies provide a vital resource that enables the use of LCLs in functional cell and molecular biology experiments. Potential applications include the characterisation of genetic variants of unknown significance, the interrogation of cellular disease pathways and mechanisms, and high-throughput discovery of genetic modifiers of disease states among others.|
|Keywords:||Lymphocytes; Cell Line; Humans; Lentivirus; Transduction, Genetic; Genetic Vectors; Female; Male|
|Description:||Published online: 20 June 2018|
|Rights:||© The Author(s) under exclusive licence to The Japan Society of Human Genetics 2018|
|Appears in Collections:||Molecular and Biomedical Science publications|
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