Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency
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)
- et al.
Exp. Cell Res.
(1997) - et al.
Neuroscience
(2002) - et al.
Nature
(1981) Proc. Natl. Acad. Sci. USA
(1981)- et al.
Science
(1998) - et al.
Proc. Natl. Acad. Sci. USA
(1998) - et al.
Proc. Natl. Acad. Sci. USA
(1988) - et al.
Methods Mol. Biol.
(1995) - et al.
Gene Ther.
(1997) - et al.
Curr. Pharm. Biotechnol.
(2001)
Cited by (18)
Application of Embryonic Stem Cells on Parkinson's Disease Therapy
2011, Genomic Medicine, Biomarkers, and Health SciencesSox2 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 CommunicationsCitation 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, BiomaterialsCitation 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.
Transduction of murine embryonic stem cells by magnetic nanoparticle-assisted lentiviral gene transfer
2013, Methods in Molecular BiologyGreen fluorescent protein as indicator of nonviral transient transfection efficiency in endometrial and testicular biopsies
2010, Microscopy Research and Technique