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. 2014 Jun;10(3):408-16.
doi: 10.1007/s12015-014-9500-9.

Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation

Nibedita Lenka  1 , Shruthi Krishnan, Philip Board, Danny Rangasamy
Affiliations

Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation

Nibedita Lenka et al. Stem Cell Rev Rep. 2014 Jun.

Abstract

Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

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Conflict of interest statement

No potential competing interests were disclosed.

Figures

Fig. 1
Fig. 1
Outline of the steps involved in L1-mediated loss-of-function screens to find genes associated with stem cell differentiation and self-renewal
Fig. 2
Fig. 2
Schematic diagrams of the pL1-EGFP-S/MAR retrotransposon vector system. GFP is co-expressed as a single fusion transcript due to the presence of the splicing sites in the intron sequences. This arrangement ensures that GFP expression occurs only after successful insertion of an L1 copy into a gene. The vector map (left panel) and the flowchart of L1 retrotransposition event or gene disruption (right panel) are shown. SD splice donor; SA splice acceptor; CMV cytomegalovirus early promoter; S/MAR scaffold/matrix attachment regions; GFP GFP reporter gene
Fig. 3
Fig. 3
Generation of loss-of-function screens using the L1 retrotransposon system. Mouse ESCs are transfected with L1 vector and allowed to proliferate for 7 days in the presence of LIF to initiate gene disruption. a A representative result of ESC colonies expressing GFP and no GFP expression formed by transfected cells. b Rationale for the PCR analysis. The 1.5 kb PCR product represents the original L1 vector harboring the intron-containing GFP, while 0.5 kb PCR product indicates a gene disruption and the loss of the intron. c A representative result of ESCs analyzed by PCR amplification. The symbol – and + represents the GFP-positive and GFP-negative ESC colonies. Negative, untransfected (control) ESCs; Marker, 1 kb-plus DNA marker
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