Elsevier

Methods

Volume 33, Issue 2, June 2004, Pages 113-120
Methods

Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency

https://doi.org/10.1016/j.ymeth.2003.11.008Get rights and content

Abstract

Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 μg DNA for 10 × 106 cells via electroporation which resulted in 10–20 positive cells/mm2, M9-assisted lipofection of 2 × 105 cells with 2 μg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 × 106 ES cells and 6 μg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.

Introduction

Pluripotent embryonic stem (ES) cells, derived from the embryo blastocyst inner cell mass, can be propagated in an undifferentiated state in vitro. Mouse ES (mES) cells were first isolated in the early 1980s [1], [2] while human ES cells and human embryonic germ cells (EG cells) were isolated in 1998 [3], [4]. ES cells are a major tool in knock-out mouse technologies, tissue engineering applications, developmental biology, and differentiation studies. ES cells also serve as a potential resource for gene therapy and cell therapy. Genetically engineered mice have become invaluable biological tools for investigating gene function and disease pathogenesis. With the human and mouse genome projects completed or near completion, the demand for large numbers of mouse models bearing predetermined genetic alterations obtained via ES cell technology is higher than ever.

ES cells retain their pluripotency for a limited time in culture and are extremely difficult to lipofect. Most gene targeting methods utilize inefficient, expensive, and time consuming electroporation approaches to introduce foreign genes into ES cells [5], [6], [7]. Electroporation is typically used to transfect mES cells, although the rate of mES cell survival following electroporation can be as low as 10% [5]. Electroporation may be particularly problematic for human ES cell applications where survivability of rare human stem cells, such as those from autologous cord blood, is critical. Electroporation is also labor intensive since it requires millions of cells and large quantities of plasmid (a maxiprep) [5]. A simple and efficient lipofection approach for mES transfection would be valuable for ES cell techniques and developmental studies.

Lipofection has low efficiency in certain cell types due to intracellular barriers, such as poor endocytosis, poor endosome escape, and/or poor nuclear localization of the transfected DNA [8]. ES cells are notoriously difficult to lipofect, and most commercially available lipid-based transfection reagents have either low transfection efficiency or high toxicity when used with mES cells [9]. We sought to address this problem by testing a nonclassical nuclear localization (NLS) peptide to assist in lipofection.

Nuclear import is believed to be a rate-limiting step during gene delivery. In an early micro-injection study, Capecchi [10] showed that less than 1% of cells expressed transgenes when plasmid was injected into cytoplasm, while up to 50% cells expressed transgenes when plasmid was injected into the nucleus directly. Classical nuclear localization signals (cNLS) have been tested, but result in only a modest or negligible increase in transfection efficiency in commonly used cell lines, such as HeLa and 3T3 cells [11], [12], [13]. To overcome these problems, we turned to a nonclassical NLS termed M9, which is derived from the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 [14], [15]. By conjugating M9 with a cationic peptide sequence for DNA binding, transgene expression increased 63-fold in confluent bovine aortic endothelium cells [16].

In this study, we tested the use of the M9 sequence for lipofection of mES cells. Our results demonstrated that M9-assisted lipofection dramatically increased transgene expression in mES cells. In addition, mES transfected with M9-lipofection retain their pluripotency and can contribute to the germline. This simple transfection method provides an alternative to electroporation for introducing DNA into ES cells. To our knowledge, this is the first report of transgenic or knock-in mouse generation from lipofected cells. Such a method may facilitate efforts to create large numbers of mouse models for genomic investigations of gene function and disease pathogenesis.

Section snippets

Transfection efficiency of electroporation and lipofection

To compare the transfection efficiency of conventional electroporation and liposome-based transfection, we transfected AB2.2 mES cells with the pcDNA3-EGFP plasmid. Electroporation was performed as described in Section 4, using 10 × 106 cells and 20 μg plasmid DNA. Fluorescence microscopy performed 48 h after electroporation demonstrated less than 10–20 weakly fluorescent EGFP positive cells/mm2 (Fig. 1A). FACS analysis of the same mES cells showed that 4.8% of electroporated mES cells had

Discussion

ES cells are important resources for both fundamental research and potential therapeutic applications. The introduction of genes into ES cells is an important tool for analysis of DNA function, cell differentiation, cell/gene therapy, tissue engineering, and knock-out production [17], [18]. We have demonstrated that M9-assisted lipofection of mES cells has higher transfection efficiency than electroporation and that this simple technique saves time and materials when used for gene targeting of

Cell culture

AB2.2 mouse embryonic stem cells and ESQ feeder cells were purchased from Stratagene. STO feeder cells were obtained from ATCC. Feeder cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) with 7–10% fetal bovine serum (FBS) (Hyclone). ES cells were cultured on Mitomycin C (10 μg/ml) inactivated feeders in ES medium (DMEM with 15% FBS, 1% nonessential amino acid (Gibco-BRL), 0.1 mM β-mercaptoethanol (Sigma), and 1250 U/ml leukemia inhibitory factor (LIF) (Chemicon)). For use in

Acknowledgments

The authors thank Dr. Romaica Omarrudin and Mr. David Pugh for their expert technical assistance, and are grateful to Dr. Jean Richa and other members of the Transgenic and Chimeric Mouse Core Facility for their assistance. This work was supported by Grants from the National Institutes of Health (RO3 EY013776 to E.A.P., RO1 HL66565 to S.L.D., and PO1-CA072765-06A1), the Rosanne H. Silbermann Foundation, the Mackall Foundation Trust, and the F.M. Kirby Foundation. H.M. is an NIH postdoctoral

References (21)

  • M. Watanabe et al.

    Exp. Cell Res.

    (1997)
  • H. Ma et al.

    Neuroscience

    (2002)
  • M.J. Evans et al.

    Nature

    (1981)
  • G.R. Martin

    Proc. Natl. Acad. Sci. USA

    (1981)
  • J.A. Thomson et al.

    Science

    (1998)
  • M.J. Shamblott et al.

    Proc. Natl. Acad. Sci. USA

    (1998)
  • T. Doetschman et al.

    Proc. Natl. Acad. Sci. USA

    (1988)
  • J.S. Mudgett et al.

    Methods Mol. Biol.

    (1995)
  • N.S. Templeton et al.

    Gene Ther.

    (1997)
  • H. Ma et al.

    Curr. Pharm. Biotechnol.

    (2001)
There are more references available in the full text version of this article.

Cited by (18)

  • Application of Embryonic Stem Cells on Parkinson's Disease Therapy

    2011, Genomic Medicine, Biomarkers, and Health Sciences
  • Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells

    2010, Biochemical and Biophysical Research Communications
    Citation Excerpt :

    Thus it is suggested that SRR2-DTS works as an ES cell-specific DTS. Despite of safety advantages of non-viral methods, existing methods do not have enough transfection efficiency, and thus many approaches including the improvement of additional factors have been explored for gene delivery to ES cells [26,27]. Utilizing a DTS, the simple insertion of specific DNA sequence into plasmids, offers another approach which may be able to combine existing methods to achieve an effective gene delivery.

  • Microsphere-based tracing and molecular delivery in embryonic stem cells

    2009, Biomaterials
    Citation Excerpt :

    Thus in parallel with bead treatment, all cell types were transfected with a plasmid expressing EGFP under optimal conditions. As observed by others [4,6,7], the transfection efficiency achieved in undifferentiated ES cultures by Lipofectamine was at best 40% (Fig. 2A). While the efficiency of microsphere uptake may be high, its effectiveness as a biological delivery vehicle for proteins and RNAi depends on subcellular localization of internalized microspheres.

View all citing articles on Scopus
View full text