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.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Neitzel H. A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet. 1986;73:320–6.
Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 2004;4:757–68.
Hussain T, Mulherkar R. Lymphoblastoid cell lines: a continuous in vitro source of cells to study carcinogen sensitivity and DNA repair. Int J Mol Cell Med. 2012;1:75–87.
Mohyuddin A, Ayub Q, Siddiqi S, Carvalho-Silva DR, Mazhar K, Rehman S, et al. Genetic instability in EBV-transformed lymphoblastoid cell lines. Biochim Biophys Acta. 2004;1670:81–83.
Sie L, Loong S, Tan EK. Utility of lymphoblastoid cell lines. J Neurosci Res. 2009;87:1953–9.
International HapMap C, Altshuler DM, Gibbs RA, Peltonen L, Altshuler DM, Gibbs RA, et al. Integrating common and rare genetic variation in diverse human populations. Nature. 2010;467:52–8.
Genomes Project C, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, et al. A global reference for human genetic variation. Nature. 2015;526:68–74.
Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489:57–74.
Wheeler HE, Dolan ME. Lymphoblastoid cell lines in pharmacogenomic discovery and clinical translation. Pharmacogenomics. 2012;13:55–70.
Caron M, Imam-Sghiouar N, Poirier F, Le Caer JP, Labas V, Joubert-Caron R. Proteomic map and database of lymphoblastoid proteins. J Chromatogr B Anal Technol Biomed Life Sci. 2002;771:197–209.
Dirksen EH, Cloos J, Braakhuis BJ, Brakenhoff RH, Heck AJ, Slijper M. Human lymphoblastoid proteome analysis reveals a role for the inhibitor of acetyltransferases complex in DNA double-strand break response. Cancer Res. 2006;66:1473–80.
Wishart DS, Knox C, Guo AC, Eisner R, Young N, Gautam B, et al. HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res. 2009;37:D603–10.
Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, et al. HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007;35:D521–26.
Lappalainen T, Sammeth M, Friedlander MR, t Hoen PA, Monlong J, Rivas MA, et al. Transcriptome and genome sequencing uncovers functional variation in humans. Nature. 2013;501:506–11.
Mele M, Ferreira PG, Reverter F, DeLuca DS, Monlong J, Sammeth M, et al. Human genomics. The human transcriptome across tissues and individuals. Science. 2015;348:660–5.
Lim ET, Wurtz P, Havulinna AS, Palta P, Tukiainen T, Rehnstrom K, et al. Distribution and medical impact of loss-of-function variants in the Finnish founder population. PLoS Genet. 2014;10:e1004494.
Gamazon ER, Duan S, Zhang W, Huang RS, Kistner EO, Dolan ME, et al. PACdb: a database for cell-based pharmacogenomics. Pharm Genom. 2010;20:269–73.
Min JL, Taylor JM, Richards JB, Watts T, Pettersson FH, Broxholme J, et al. The use of genome-wide eQTL associations in lymphoblastoid cell lines to identify novel genetic pathways involved in complex traits. PLoS ONE. 2011;6:e22070.
Welsh M, Mangravite L, Medina MW, Tantisira K, Zhang W, Huang RS, et al. Pharmacogenomic discovery using cell-based models. Pharmacol Rev. 2009;61:413–29.
Izawa K, Martin E, Soudais C, Bruneau J, Boutboul D, Rodriguez R, et al. Inherited CD70 deficiency in humans reveals a critical role for the CD70-CD27 pathway in immunity to Epstein-Barr virus infection. J Exp Med. 2017;214:73–89.
Jiang S, Zhou H, Liang J, Gerdt C, Wang C, Ke L, et al. The Epstein-Barr virus regulome in lymphoblastoid cells. Cell Host Microbe. 2017;22:561–73. e564
Ma Y, Walsh MJ, Bernhardt K, Ashbaugh CW, Trudeau SJ, Ashbaugh IY, et al. CRISPR/Cas9 screens reveal epstein-Barr virus-transformed B cell host dependency factors. Cell Host Microbe. 2017;21:580–91. e587
Chandra S, Levran O, Jurickova I, Maas C, Kapur R, Schindler D, et al. A rapid method for retrovirus-mediated identification of complementation groups in Fanconi anemia patients. Mol Ther. 2005;12:976–84.
Muller LU, Milsom MD, Kim MO, Schambach A, Schuesler T, Williams DA. Rapid lentiviral transduction preserves the engraftment potential of Fanca-/- hematopoietic stem cells. Mol Ther. 2008;16:1154–60.
Portal D, Zhao B, Calderwood MA, Sommermann T, Johannsen E, Kieff E. EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding. Proc Natl Acad Sci USA. 2011;108:7808–13.
Sommermann TG, O’Neill K, Plas DR, Cahir-McFarland E. IKKbeta and NF-kappaB transcription govern lymphoma cell survival through AKT-induced plasma membrane trafficking of GLUT1. Cancer Res. 2011;71:7291–300.
Wang X, Berger C, Wong CW, Forman SJ, Riddell SR, Jensen MC. Engraftment of human central memory-derived effector CD8 +T cells in immunodeficient mice. Blood. 2011;117:1888–98.
Arvey A, Tempera I, Tsai K, Chen HS, Tikhmyanova N, Klichinsky M, et al. An atlas of the Epstein-Barr virus transcriptome and epigenome reveals host-virus regulatory interactions. Cell Host Microbe. 2012;12:233–45.
White RE, Ramer PC, Naresh KN, Meixlsperger S, Pinaud L, Rooney C, et al. EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors. J Clin Invest. 2012;122:1487–502.
Banzhaf-Strathmann J, Claus R, Mucke O, Rentzsch K, van der Zee J, Engelborghs S, et al. Promoter DNA methylation regulates progranulin expression and is altered in FTLD. Acta Neuropathol Commun. 2013;1:16.
Zhao B, Barrera LA, Ersing I, Willox B, Schmidt SC, Greenfeld H, et al. The NF-kappaB genomic landscape in lymphoblastoid B cells. Cell Rep. 2014;8:1595–606.
Greenfeld H, Takasaki K, Walsh MJ, Ersing I, Bernhardt K, Ma Y, et al. TRAF1 coordinates polyubiquitin signaling to enhance Epstein-Barr virus LMP1-mediated growth and survival pathway activation. PLoS Pathog. 2015;11:e1004890.
Ohashi M, Holthaus AM, Calderwood MA, Lai CY, Krastins B, Sarracino D, et al. The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth. PLoS Pathog. 2015;11:e1004822.
Nguyen LS, Jolly L, Shoubridge C, Chan WK, Huang L, Laumonnier F, et al. Transcriptome profiling of UPF3B/NMD-deficient lymphoblastoid cells from patients with various forms of intellectual disability. Mol Psychiatry. 2012;17:1103–15.
Jolly LA, Homan CC, Jacob R, Barry S, Gecz J. The UPF3B gene, implicated in intellectual disability, autism, ADHD and childhood onset schizophrenia regulates neural progenitor cell behaviour and neuronal outgrowth. Hum Mol Genet. 2013;22:4673–87.
Weber K, Bartsch U, Stocking C, Fehse B. A multicolor panel of novel lentiviral “gene ontology” (LeGO) vectors for functional gene analysis. Mol Ther. 2008;16:698–706.
Weber K, Mock U, Petrowitz B, Bartsch U, Fehse B. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis. Gene Ther. 2010;17:511–20.
Koh CM. Preparation of cells for microscopy using cytospin. Methods Enzymol. 2013;533:235–40.
Kumar R, Corbett MA, van Bon BW, Woenig JA, Weir L, Douglas E, et al. THOC2 mutations implicate mRNA-export pathway in X-linked intellectual disability. Am J Hum Genet. 2015;97:302–10.
Corbett MA, Schwake M, Bahlo M, Dibbens LM, Lin M, Gandolfo LC, et al. A mutation in the Golgi Qb-SNARE gene GOSR2 causes progressive myoclonus epilepsy with early ataxia. Am J Hum Genet. 2011;88:657–63.
Maruo S, Wu Y, Ishikawa S, Kanda T, Iwakiri D, Takada K. Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells. Proc Natl Acad Sci USA. 2006;103:19500–5.
Weber K, Thomaschewski M, Benten D, Fehse B. RGB marking with lentiviral vectors for multicolor clonal cell tracking. Nat Protoc. 2012;7:839–49.
Weber K, Thomaschewski M, Warlich M, Volz T, Cornils K, Niebuhr B, et al. RGB marking facilitates multicolor clonal cell tracking. Nat Med. 2011;17:504–9.
Bell JT, Pai AA, Pickrell JK, Gaffney DJ, Pique-Regi R, Degner JF, et al. DNA methylation patterns associate with genetic and gene expression variation in HapMap cell lines. Genome Biol. 2011;12:R10.
Consortium GT. Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science. 2015;348:648–60.
Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res. 2014;42:e147.
Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol. 2014;32:267–73.
Boutros M, Ahringer J. The art and design of genetic screens: RNA interference. Nat Rev Genet. 2008;9:554–66.
Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32:347–55.
Acknowledgements
L.A.J. is supported by Australian Research Council DE160100620. J.G. is supported by National Health and Medical Research Council (NHMRC) of Australia grants 1041920 and 1091593. This work was supported by NHMRC GNT1063808 to J.G. and L.A.J.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
This Research was prospectively reviewed and approved by the Women’s and Children’s Hospital Human Research Ethics Committee, South Australia, Australia, 5006. Informed consent was obtained from all individual participants included in the study. The authors declare that they have no conflict of interest.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Jolly, L.A., Sun, Y., Carroll, R. et al. Robust imaging and gene delivery to study human lymphoblastoid cell lines. J Hum Genet 63, 945–955 (2018). https://doi.org/10.1038/s10038-018-0483-2
Received:
Revised:
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s10038-018-0483-2
