Generation of conditional MEN1 knockout human induced pluripotent stem cells (iPSCs) provide a genetically-tractable disease model to investigate cell-type specific gene function

Research output: Contribution to journalConference articlepeer-review

Abstract

Introduction: Germline and somatic inactivating MEN1 variants are associated with a wide range of inherited and sporadic endocrine tumours. MEN1 encodes the tumour suppressor Menin, a ubiquitously expressed scaffold protein, implicated in multiple cellular processes including transcription, epigenetic regulation, and modulation of key signaling pathways. Despite intensive study, the mechanisms leading to endocrine tumorigenesis remain ill-defined, in part reflecting a lack of physiologically relevant model systems. Here, applying a methodology known as CRISPR-FLIP, we report a conditional MEN1 knockout human iPSC line, offering a novel platform to investigate cell-type specific gene function.

Methods: A targeting strategy was designed to introduce an invertible ‘FLIP’ cassette into exon 2 of the MEN1 gene. Appropriate gRNA/Cas9 and custom-designed donor repair template (containing the FLIP cassette) vectors were generated and electroporated into the ChiPS4 cell line. Successfully targeted cells were then transfected with a piggyBac-GFP ERT2-Cre-ERT2 vector. GFP-expressing cells (indicating stable incorporation of ERT2-Cre-recombinase) were isolated, expanded and assessed for maintained pluripotency.

Results: Clonal cell populations with bi-allelic integration of the FLIP cassette within the MEN1 gene and stable integration of ERT2-Cre recombinase were successfully generated. Maintained pluripotency was confirmed by retained expression of pluripotency markers and embryoid body formation. Confirming the utility of the cell model, treatment of undifferentiated cells with tamoxifen resulted in undetectable Menin expression after 5-days as assessed by Western blot and immunocytochemistry. Subsequently, the consequence of Menin depletion during differentiation was evaluated using RNA sequencing of tamoxifen-treated pancreatic endoderm cells (PDX1/SOX9 positive) undergoing differentiation to pancreatic progenitors (PDX1/NKX6.1 positive), identifying alterations in multiple genes and pathways relevant to endocrine development and tumorigenesis.

Discussion: Conditional MEN1 knockout human iPSCs provide a powerful model to investigate gene function in physiologically-relevant cellular environments, thereby providing a tool that can offer novel insights into tumour biology and may be exploited for drug discovery.
Original languageEnglish
JournalEndocrine Abstracts
Volume86
DOIs
Publication statusPublished - Nov 2022
EventSociety for Endocrinology BES 2022 - Harrogate, United Kingdom
Duration: 14 Nov 202216 Nov 2022

Fingerprint

Dive into the research topics of 'Generation of conditional MEN1 knockout human induced pluripotent stem cells (iPSCs) provide a genetically-tractable disease model to investigate cell-type specific gene function'. Together they form a unique fingerprint.

Cite this