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江边孤钓

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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

a Department of Bioscience, National Cardiovascular Center Research Institute;4 {; D8 ~# x- U" E. G
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b Department of Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Suita, Osaka, Japan

Key Words. Embryonic stem cells ? Green fluorescent protein ? Small interfering RNA ? Homologous recombination ? Hprt locus ? Flow cytometry' ?! C- Q! W4 I1 ^/ W% i
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Correspondence: Takayuki Morisaki, M.D., Ph.D., Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Telephone: 81-6-6833-5012, ext. 2506; Fax: 81-6-6835-5451; e-mail: morisaki@ri.ncvc.go.jp, v# a& J5 V6 G. r

ABSTRACT* q5 I" M7 L8 W2 p

Embryonic stem cells (ESCs) are pluripotent cell lines derived from the inner cell mass of blastocyst-stage embryos that have been shown to differentiate in vivo and in vitro into all cell lineages of adult animals. Although there is considerable interest in the therapeutic applications of ESCs , they cannot be used in regenerative therapies in an undifferentiated state, because they form teratomas after transplantation . Hence, a thorough understanding of the process of lineage commitment and differentiation of ESCs is essential for developing transplantation strategies .3 I3 g; G$ B3 v% l
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To understand the specific signaling pathways involved in specification and differentiation of ESC-derived cells, it is important to elucidate the function of specific genes by manipulating their level of expression . In mouse studies, homologous recombination–based gene knockout methods have been shown to be powerful tools to understand the function of genes, and several reports have demonstrated that knockout of developmentally important genes has an effect on differentiation ability in vitro . However, knockout methods are used infrequently in in vitro studies because an additional round of transfection is required to knock out all of the alleles.
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RNA interference (RNAi) is a recently elucidated technique that has been used successfully to suppress gene expression in Caenorhabiditis elegans and Drosophila melanogaster. In mammalian cells, including mouse and human ESCs, it was recently shown that small interfering RNA (siRNA), administered as a 21-nt double-stranded RNA, could suppress gene expression, though the knockdown effect was transient and could be maintained only for a short term . Very recently, a hairpin siRNA expression system driven by the RNA polymerase III promoter was shown to be able to suppress target gene expression in ESCs . This vector-based technology is a simple and promising means for exploring the biological functions of differentiation-related genes in ESCs, whereas the availability of an RNAi technique for ESCs also provides a convenient method for directly generating knockdown mice, by utilizing tetraploid embryo . In the present study, we found that an siRNA expression cassette, site-specifically integrated into the constitutively active hypoxanthine guanine phosphoribosyl transferase 1 (Hprt) locus, uniformly suppressed target gene expression over a long term. As speculated, the effect of gene suppression was highly reproducible and persisted throughout the process of differentiation by ESCs.* f7 ]  i1 C" H1 L/ h5 y
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MATERIALS AND METHODS
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Site-Directed Integration of siRNA Expression Cassette at Hprt Locus
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Constitutively active housekeeping gene loci, such as the Hprt locus and ROSA26 locus, are often used to introduce transgenesis in murine ESCs by homologous recombination . To use the Hprt locus, we deleted the first exon of the Hprt gene, using an Hprt-deleting vector by homologous recombination, as described previously . We also inserted the EGFP reporter gene into the ROSA26 locus to ensure reporter expression over a long term . The resultant Hprt–EGFP  ESC line (termed hgh2; Fig. 1A), was then transfected with the Hprt targeting vector (pHprt-siGFP; Fig. 1B), which contained the human H1-promoter–driven siRNA expression cassette for the EGFP gene. Because the Hprt targeting vector carries the first and second exons of the Hprt gene, only homologous recombined clones were rescued and became Hprt  (Fig. 1C). Among the HAT-resistant colonies that appeared on day 10, nearly all exhibited much less fluorescence as compared with the hgh2 cells, as assessed under a fluorescence microscope (Fig. 2A). Five randomly selected clones were found to contain only one copy of the human H1 promoter sequence, as determined by quantitative PCR of genomic DNA (data not shown), suggesting that the siRNA cassette was solely integrated into the Hprt locus and not integrated randomly into the genome. As shown in Figure 2B, these five clones expressed approximately 30-fold lower GFP fluorescence than the hgh2 cells, suggesting that suppression by the siRNA cassette integrated at the Hprt locus was highly reproducible and homogeneous.) @  k& a; {4 q
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Figure 1. Introduction of siRNA expression cassette at the Hprt locus. (A): The Hprt–EGFP  cell line, hgh2, is depicted. It constitutively expresses EGFP driven by the CAG promoter at the ROSA26 locus. In this cell line, the genomic region containing the first exon of the Hprt gene was replaced with a neomycin-resistant gene cassette (MC1neo). (B): Schematic design of the Hprt targeting vector containing the first and second exons of the Hprt gene is shown. The red box indicates the siRNA expression cassette, containing the H1 promoter and the short hairpin small interfering sequence. The short hairpin sequence for EGFP, indicated in the lower part, is derived from pGtoR. (C): The siRNA-expressing ESC derived from hgh2 is depicted. By homologous recombination with the Hprt targeting vector, the Hprt locus is reconstituted to confer Hprt positivity and the siRNA cassette for GFP is integrated at the Hprt locus. Abbreviations: EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; GFP, green fluorescent protein; siRNA, small interfering RNA.( m% R0 K$ ?: }3 c; l

Figure 2. Long-term suppression of EGFP in five in dependent clones. (A): Fluorescence of siGFP-expressing ESCs. The Hprt–EGFP  ESC line, hgh2, was transfected with the Hprt targeting vector containing siRNA cassette for EGFP. Nearly all Hprt  clones exhibited reduced expression of EGFP. One of the siGFP-expressing clones (#1), hgh2, and its parental strain, ht7, are shown. (B): Relative intensity of GFP fluorescence of siGFP-expressing ESCs (#1–#5) before (passage 1) and after (passage 10) long-term culture. Mean fluorescence intensity of hgh2 obtained from flow cytometry analysis is set as 100%. Abbreviations: EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; GFP, green fluorescent protein; PI, propidium iodide; siGFP, small interfering RNA–expressing cassette for GFP; siRNA, small interfering RNA.

To test the stability of the suppression effect, we cultured ESCs expressing siRNA for 10 passages without HAT selection and analyzed GFP fluorescence by flow cytometry. As indicated in Figure 2B, a suppressing effect was observed after approximately 1 month without any significant decrease, indicating that the expression of the siRNA cassette from the Hprt locus was highly stable and not subject to an epigenetic effect.
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Gene Suppression in Differentiated Cells Derived from ESCs7 f/ `- |2 s* M, X
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The siGFP-expressing ESCs underwent multiple rounds of transfection; therefore, it was considered important to determine if they retained the ability to differentiate into multiple lineages of cells. The siGFP-expressing ESCs formed EBs as efficiently as the control ESC clones. Furthermore, the efficiency for production of EBs that exhibited spontaneous beating was nearly comparable to that of the original cell line, ht7 (Fig. 3A), indicating that the cardiomyogenic potential was retained. We also observed multiple cell types, such as smooth muscle, cardiac muscle (Fig. 3B), endothelial-like, and neuron-like cells (data not shown) that were derived from the siGFP-expressing ESCs. Thus, the siGFP-expressing ESCs retained a differentiation potential comparable to that of the original ESCs.
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Figure 3. Differentiation of EBs derived from stably transfected cells. (A): Frequency of spontaneous beating EBs derived from siGFP-expressing ESCs. EBs were formed by the hanging drop method. On day 5 of differentiation, EBs were transferred onto 24-well gelatin coated plates and the number of spontaneous beating EBs was counted. Three independent differentiation experiments were performed for each clone. (B): Differentiation of siGFP-expressing cells into sarcomeric myosin heavy chain–positive or SM actin–positive cells. The siGFP-expressing EBs were dissociated and replated onto a gelatin-coated 35-mm dish. Immunocytochemical analysis was performed using anti-sarcomeric myosin heavy chain antibody (MF20; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww) and anti-SM--actin antibody (1A4; Sigma, St. Louis, http://www.sigmaaldrich.com) after incubation with the secondary antibody conjugated with Alexa546 (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com). Abbreviations: EB, embryoid body; ESC, embryonic stem cell; GFP, green fluorescent protein; siGFP, small interfering RNA–expressing cassette for GFP; SM, smooth muscle.

Those cells differentiated from EBs that were derived from siGFP-expressing cells exhibited a much weaker fluorescence than cells derived from the parental ESCs (hgh2), as assessed with a fluorescence microscope (Fig. 4A). To quantify the suppressive effect of RNAi in the differentiated cells, EBs derived from five independent clones were dissociated and analyzed by flow cytometry. On average, the siGFP-expressing ESC-derived cells exhibited an approximately 10-fold lower level of fluorescence than hgh2 cells (Fig. 4B). Furthermore, there were no significant differences in the levels of suppression among the individual clones, whereas each exhibited a similar GFP fluorescence histogram profile (data not shown). The histogram pattern of each clone also suggested that RNAi occurred uniformly in each differentiated cell, though not in specific patterns in individual cell types. These results suggested that the suppression of GFP by the siRNA cassette was equally effective in each stage of differentiation of ESCs." v5 M" l4 j4 b, Y/ m$ {5 j) x

Figure 4. Gene suppression of GFP in differentiated cells. (A): Fluorescence of EBs derived from siGFP-expressing ESCs. EBs were formed as described in Results. GFP fluorescence was observed under a fluorescence microscope. (B): Relative intensity of GFP fluorescence of siGFP-expressing ESC-derived cells. Mean fluorescence of hgh2 obtained from flow cytometry analysis is set as 100%. Abbreviations: EB, embryoid body; ESC, embryonic stem cell; GFP, green fluorescent protein; siGFP, smal interfering RNA–expressing cassette for GFP.3 I/ \1 v5 F& U8 _7 f" i9 {
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Suppression of Endogenous Genes by siRNA Cassette Integrated at the Hprt Locus
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To test if the siRNA expression system also functions to suppress endogenous genes in ESCs, we designed three different siRNA cassettes for the Flk1 (Kdr) gene (siFlk1-A, -B, and -C) and integrated them into the Hprt locus of Hprt-ESCs (hdh31) using the Hprt targeting vector. The Flk1 gene encodes vascular endothelial growth factor receptor 2 and is expressed in nascent mesoderm, hemangioblast, and endothelial cells derived from ESCs . Typically, more than 30% of the ESCs derived from wild-type ESCs (ht7) became Flk1-positive on day 5 (Fig. 5A). Whereas one of the siRNA cassettes (siFlk1-A) did not seem to be effective in suppression of the Flk1 gene, the two others (siFlk1-B and -C) efficiently suppressed its expression. As shown in Figure 5B, only 2%–4% of the cells expressing siFlk1-B or -C were Flk1-positive. The Flk1-positive cells derived from siRNA-expressing ESCs exhibited weaker fluorescence as compared with cells derived from wild-type ESCs (approximately 50% reduction for siFlk-B or -C). These results suggest that siRNA from the Hprt locus can suppress endogenous genes efficiently and stably in ESCs.
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Figure 5. Gene suppression of Flk1 in differentiated cells. (A): Flow cytometry analysis of siFlk1-expressing ESC-derived EBs. The ht7-derived Hprt– ESC line, hdh31, was transfected with the Hprt targeting vectors with three different designs of siRNA cassette for Flk1 (siFlk1-A, -B, and -C). The Hprt  clones were selected, expanded, and differentiated by the method of EB formation. EBs were dissociated by trypsin-EDTA on day 5, stained with anti-Flk1 antibody conjugated with PE. Dead cells were stained with 7AAD. Flow cytometry analyses of ht7– and siFlk1-B–expressing cells are shown in the left and right frames, respectively. (B): Frequency of Flk1-positive cells in ht7- and siFlk1-expressing EBs (left). Two independent clones for each siFlk1 cassette (siFlk1-A, -B, and -C) were examined, and similar results were obtained (data not shown). Relative intensity of PE fluorescence in the Flk1-positive cell fraction of siFlk1-expressing EBs (right). Mean fluorescence of Flk1-PE–positive cell fraction (R2 in Figure 5A) of ht7 is set as 100%. Abbreviations: 7AAD, 7-amino-actinomycin D; EB, embryoid body; ESC, embryonic stem cell; PE, phycoerythrin; siRNA, small interfering RNA." x, m6 H- ]( x" J

DISCUSSION

We express our thanks to H. Niwa for providing the ht7 ESCs, S.K. Bronson for plasmid pHprt, S. Duncan for plasmid pMP8NEBlacZ, M. Okabe for plasmid pGtoR, J. Miyazaki for plasmid pCX-EGFP, and P. Soriano for plasmid pROSA26-1. This work was supported in part by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan, by Research Grants on Cardiovascular Diseases from the Ministry of Health, Labor, and Welfare, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences (JSPS).1 G$ J$ L7 M  O+ k) p# \' u

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