Conditional Gene Expression in Human Embryonic Stem Cells



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发表于 2009-3-5 00:53 |显示全部帖子

作者：Ludovic Vallier, Morgan Alexander, Roger Pedersen作者单位：Department of Surgery and Cambridge Institute for Medical Research, Addenbrooke   N+ K! }6 K4 M6 Z' i

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      【摘要】
   Human embryonic stem cells (hESCs) possess unique properties for studying mechanisms controlling cell fate commitment during early mammalian development. Gain of function is a common strategy to study the function of specific genes involved in these mechanisms. However, transgene toxicity can be a major limitation, especially with factors influencing proliferation or differentiation. Here, we describe an efficient method based on the inducible recombinase Cre-ERT2 for conditional gene expression in hESCs and their differentiated derivatives. Using this approach, we have established several hESC sublines inducible for the expression of the enhanced green fluorescent protein and the transforming growth factor ¦Â family member Nodal. Together, these results demonstrate that Cre-ERT2 can be used to control gene expression in undifferentiated and differentiated cells, thereby providing the first conditional transgene expression system that works effectively in hESCs.
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Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Human embryonic stem cells Cre recombinase Tamoxifen Nodal Inducible expression Pluripotency, u$ g+ M3 p0 o2 G- k' P: C4 h  ?' x: i
   INTRODUCTION

Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of human embryos at the blastocyst stage . Therefore, the Cre-ERT2 system can be efficiently used to control gene expression in hESCs and their differentiated progeny, thereby providing the first conditional system of expression available in this key pluripotent cell system.3 t/ C9 v% L+ A* u6 e

Figure 1. Toxicity of Cre-ERT2 induction by OHT on human ESCs (hESCs). (A): Maps of the Cre-ERT2pTP6, ZEG, and ZNO vectors. pTP6 vector contains the CAGG promoter followed by a gene of interest (here, Cre-ERT2) and an IRES-separated puromycin resistance gene allowing strong selection for transgene expression. ZEG and ZNO vectors have CAGG-regulated expression of eGFP or Nodal, which is blocked by the Cre-excisable ¦ÂGeo cassette. (B): Cell survival after activation of Cre-ERT2 by OHT induction. Control hESCs (left panel) and hESCs-expressing Cre-ERT2 (middle and right panels) were grown in chemically defined medium in the presence of increasing doses of OHT for various periods. Surviving colonies were stained using crystal violet. Abbreviations: eGFP, enhanced green fluorescent protein; min, minutes; OHT, hydroxytamoxifen; WT, wild-type.! y- e5 I" o, q  D
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MATERIALS AND METHODS

hESC Culture and Transfection
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H9 (WiCell Research Institute, Madison, WI, http://www.wicell.org) and hSF-6 (University of California San Francisco) hESCs were cultured in chemically defined medium (CDM), supplemented with Activin (10 ng/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com, or Peprotech, Rocky Hill, NJ, http://www.peprotech.com) and fibroblast growth factor 2 (FGF2) (12 ng/ml; R&D Systems). The composition of CDM was 50% Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY, http://www.invitrogen.com) plus 50% F12 NUT-MIX (Gibco), 1% chemically defined lipids (Gibco), supplemented with 7 µg/ml insulin (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com), 15 µg/ml transferrin (Roche), 450 µM monothioglycerol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 5 mg/ml bovine serum albumin fraction V (Sigma-Aldrich). To allow hESCs to adhere in CDM, the plates were precoated with fetal bovine serum (FBS) for 24 hours at 37¡ãC and then washed twice in phosphate-buffered saline (PBS) to eliminate any serum. Every 5 days, cells were harvested using 1 mg/ml collagenase IV (Gibco) and then plated into six-well plates (Corning Costar, Acton, MA, http://www.corning.com/lifesciences) that been precoated with FBS. For stable transfection with vector encoding Cre-ERT2, one confluent well of six-well plates was plated into 12 wells precoated with FBS. After 72 hours, the cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) as described . Four days after transfection, puromycin (final concentration, 1 µg/ml) was added in CDM supplemented with Activin and FGF. Puromycin-resistant colonies that appeared by 12 days after selection were picked, dissociated, and plated onto 24-well plates precoated with FBS and then expanded for further analysis as described above.
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hESC differentiation was induced by EB formation. This was accomplished by incubating colonies in medium containing 1 mg/ml collagenase IV without FGF for 20 minutes at room temperature, after which all the colonies were detached by mechanical dissociation. The colonies were then rinsed once in CDM and grown in nonadherent conditions to generate EBs.7 |$ W) N' D  z9 Q
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Karyotype analyses were performed on H9 and hSF-6 cells at various passages, and no abnormalities were detected during 40 passages in these culture conditions.

hESC Electroporation* ?) N( t. Y( F$ O4 M/ P
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ZEG and ZNO vectors (ZNO containing Nodal coding sequence in place of the GFP cassette of the ZEG vector) were stably transfected into Cre-ERT2-expressing hESCs using electroporation, since Lipofectamine was inefficient in generating inducible clones (data not shown). Three 60-mm plates of confluent Cre-ERT2-expressing hESCs (10 x 106 cells) grown in feeder-free/serum-free conditions were incubated for 1 minute in trypsin-EDTA (Gibco) and then dissociated into a single-cell suspension. Cells were washed once in medium containing 10% FBS to inhibit the trypsin and then twice in Opti-MEM (Gibco). After the second wash, cells were resuspended in 0.8 ml of Opti-MEM in an electroporation cuvette (Bio-Rad, Hercules, CA, http://www.bio-rad.com) containing 40 µg of linearized vector and then exposed to a single 260-V, 500-mF pulse using the Bio-Rad Gene Pulser II. Cells were finally plated at high density in three wells of a six-well plate (Corning Costar) precoated with FBS. Four days after electroporation, G418 (final concentration, 200 µg/ml) was added in CDM supplemented with Activin and FGF. G418-resistant colonies that appeared within 12 days of selection were picked, dissociated, plated onto 24-well precoated FBS plates, and expanded for further analysis as described above.- ]% S/ H- r* i8 a( r) L+ G

Flow Cytometry0 F; A2 v, P3 F- ]
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For detection of the pluripotency cell surface marker Tra-1¨C60, adherent cells were washed twice in PBS and then incubated for 20 minutes at 37¡ãC in cell dissociation buffer (Gibco). Cells were then dissociated by gentle pipetting and resuspended at approximately 0.1¨C1.0 x 105 cells per milliliter in PBS 3% normal goat serum (NGS) containing 0.1% azide (Serotec Ltd., Oxford, U.K., http://www.serotec.com). Cells were incubated for 20 minutes at 4¡ãC with Tra-1-60 (1:200; Chemicon, Temecula, CA, http://www.chemicon.com) or the corresponding isotype control (mouse IgM isotype control ). Cells were then washed twice in PBS 3% NGS and incubated for 20 minutes on ice with fluorescein isothiocyanate-conjugated goat anti-mouse IgM antibody (1:200; Sigma-Aldrich). Subsequently, cells were resuspended in PBS 3% NGS for staining with 7-aminoactinomycin D (7-AAD) as a viability dye (Immunotech, Luminy, France, http://www.immunotech.com) at 20 µl/ml for 15 minutes at room temperature. Live cells identified by 7-AAD exclusion were analyzed for surface-marker expression using FACSCalibur.- E+ o2 n7 J0 T) A) y/ Z( @

RNA Extraction and Real-Time PCR3 M1 J, X# h1 W

Total RNAs were extracted from hESCs or EBs using the RNeasy Mini Kit and RNeasy Micro Kit for dissected EB layers (Qiagen, Hilden, Germany, http://www1.qiagen.com). Each sample was treated with RNase-free DNase (Qiagen) to avoid DNA contamination. A test PCR was done on all the RNA samples to verify the absence of genomic contamination. For each sample 0.5 µg of total RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen). PCR mixtures were prepared as described (Stratagene  protocol for SYBR Green real-time PCR). Real-time PCR was performed using an ABI 7700 instrument (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) with 1x Master Mix (Stratagene). Cycle conditions were as recommended by Stratagene. Sequences were as follows: PBGD-forward primer (FP), 5'-GGAGCCATGTCTGGTAACGG-3'; PBGD-backward primer (BP), 5'-CCACGCGAATCACTCTCATCT-3'; Oct4-FP, 5'-AGTGAGAGGCAACCTGGAGA-3'; Oct4-BP, 5'-ACACTCGGACCACATCCTTC-3'; Nanog-FP, 5'-CATGAGTGTGGATCCACTTG-3'; Nanog-BP, 5'-CCTGAATAAGCAGATCCATGG-3'; mouse Nodal-FP, 5'-TTCAAGCCTGTTGGGCTCTAC-3'; mouse Nodal-BP, 5'-TCCGGTCACGTCCACATCTT-3'; GFP-FP, 5'-CACATGAAGCAGCACGACTT-3'; and GFP-BP, 5'-CGTCGTCCTTGAAGAAGATGGT-3'. All the PCRs were done with a negative control containing only water (data not shown). The expression of the PBGD housekeeping gene was used to normalize PCRs.6 V( {6 g$ p4 `6 v6 R: R

Immunofluorescence& I! Q4 p7 l/ n, o7 O2 O2 Y
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hESCs were fixed for 20 minutes at 4¡ãC in 4% paraformaldehyde (PFA) and then washed three times in PBS. Cells were incubated for 20 minutes at room temperature in PBS containing 10% donkey serum (Serotec) and subsequently incubated for 2 hours at room temperature with primary antibody diluted in 1% donkey serum in PBS as follows: SSEA-4 (clone MC813, 1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww), alkaline phosphatase (Abcam, Cambridge, U.K., http://www.abcam.com), Oct-4 (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), Sox17 (R&D Systems), GATA4 (1:250; Santa Cruz), GATA6 (1:200; Abcam ). Cells were then washed three times in PBS and incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (1:200 in 1% donkey serum in PBS; Sigma-Aldrich) for 2 hours at room temperature. Unbound secondary antibody was removed by three washes in PBS. Hoechst 33258 was added to the first wash (1:10,000; Sigma-Aldrich).

¦Â-Galactosidase Staining  P1 l$ l* d, P/ Q( F! y# [# E
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hESCs were fixed for 20 minutes at 4¡ãC in 4% PFA and then washed three times for 5 minutes each time at room temperature in detergent solution (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P40 in PBS). Cells were then incubated at room temperature overnight in staining solution (0.081% K3Fe(CN)6  in detergent solution) to reveal the location of ¦Â-galactosidase activity.5 g  v, ~7 e3 l, k$ X3 P) ~/ t

RESULTS' }4 }& e# {- ]+ L# w) B+ a) H

OHT and Cre-ERT2 Toxicity
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We first determined whether OHT or Cre-ERT2 was toxic to hESCs. For OHT, this was accomplished by growing the H9 and hSF-6 hESC lines for 48 hours in feeder-free and serum-free conditions , we investigated the effect of incubating Cre-ERT2-expressing hESCs for short periods of time with increasing doses of OHT in feeder-free culture conditions (Fig. 1B). Induction of Cre-ERT2 for 15¨C30 minutes with 50 nM or 250 nM OHT either once or repeatedly over a period of several days did not induce any significant side effects, including cell death (Fig. 1B). Accordingly, Cre-ERT2 activity was induced for 15 minutes with 50 nM OHT during each of 3 consecutive days for the remainder of this study. Together, these results indicate that although constitutive recombinase Cre activity can be toxic in hESCs, this limitation can be overcome by using repetitive transitory activation.' H4 [+ Q. q/ H3 z7 ]  S/ ?, U

Generation of hESCs Conditionally Expressing the Enhanced Green Fluorescent Protein

The efficiency of the Cre-ERT2 system to control gene expression in hESCs was then evaluated by generating hESCs that were inducible for eGFP expression. For this purpose, the ZEG vector (Fig. 1A) was electroporated into Cre-ERT2-expressing hESCs, and the resulting colonies were individually picked, expanded, and screened for expression of eGFP after addition of OHT to the culture medium. The results obtained with three Cre-ERT2-expressing sublines are presented in Table 1. Induction of eGFP expression was observed in 24 of 72 of the sublines obtained. Fluorescence-activated cell sorting (FACS) analyses showed that eGFP expression could be induced after OHT treatment in 50% of the cells for 18 inducible sublines and in 75% of the cells for six inducible sublines (Fig. 2A, 2B). Induction of eGFP expression was concurrent with the disappearance of ¦ÂGeo expression, which is consistent with elimination of the ¦ÂGeo cassette as a stop sequence by recombination between the two LoxP sites (Fig. 2B). In addition, the inducible sublines lost their resistance to G418 after the OHT treatment, definitively confirming the excision of the LoxP cassette (data not shown). eGFP expression was observed in fewer than 1% of the cells in the absence of OHT (Fig. 2A, 2B), showing that background expression was very low. The level of background recombination was also assessed using real-time PCR to quantitatively detect eGFP transcripts before and after OHT treatment. These were hardly detected in the absence of Cre-ERT2 activation by OHT, whereas a 36-fold increase was observed in its presence (Fig. 3B). In addition, induction of eGFP expression did not induce a significant decrease in the total number of cells, confirming the absence of toxicity of the OHT treatment (Fig. 2C). Finally, the expression of the pluripotency markers Oct-4, SSEA-4, and alkaline phosphatase (Fig. 2D) was not altered in eGFP-expressing cells, in confirming that the pluripotency of hESCs was not affected by Cre-ERT2 activation. Alternatively, eGFP expression was induced during differentiation of EBs (Fig. 2E). For this, hESC colonies were grown in nonadherent conditions for 3 days to form EBs, and then Cre-ERT2 activity was induced for 15 minutes with 50 nM OHT during each of 3 consecutive days. FACS analyses showed that eGFP expression could be induced after OHT treatment in 65% of the cells contained in the EBs, whereas GFP was expressed by only 3% of the cells in the absence of OHT treatment (Fig. 2F). Alternatively, EBs were returned to adherent conditions for 3 days and then analyzed by immunostaining for the expression of extraembryonic markers (GATA4 and GATA6), neuroectoderm markers (Sox2, NCam, and Nestin), and mesendoderm markers (Sox17 and Brachyury) (Fig. 2G). This confirmed that eGFP expression could be induced in a large variety of cell types. Finally, similar results were obtained by OHT treatment prior to EB formation (data not shown). Together, these results demonstrate that the Cre-ERT2 system can be used efficiently to induce gene expression in hESCs without affecting their pluripotent status or broad differentiative ability.! L1 P+ e. h, Q$ Y, `9 Y

Figure 2. Inducible expression of eGFP. (A): eGFP and ¦Â-galactosidase (¦Â-gal) expression in OHT-inducible human ESCs (hESCs) before and after induction by OHT. OHT induction leads to loss of ¦Â-gal activity and gain of eGFP fluorescence. Scale bar = 200 µM. (B): Fraction of cells expressing eGFP in inducible hESCs after induction by OHT. Cre-ERT2-expressing hESCs stably transfected with the ZEG vector (ZEG 3.1) were grown in the absence (¨COHT) or presence ( OHT) of OHT, and the fraction of cells expressing GFP was then determined using fluorescence-activated cell sorting (FACS). (C): OHT treatment does not result in a significant decrease in the total number of ZEG-containing hESCs. ZEG 3.1 and ZEG 4.5 hESC lines were grown in the absence (¨COHT) or presence ( OHT) of OHT. Two days after the last OHT treatment, hESCs were incubated in trypan blue to determine the total number of living cells. OHT treatment did not affect the number of cells (ZEG 3.1, p = .2, 3 df; ZEG 4.5, p = .1, 3 df). (D): Expression of pluripotency markers after induction of eGFP expression in OHT-inducible hESCs. Expression of the pluripotency markers Oct-4 (red fluorescence, top row), AP (red fluorescence, middle row), and SSEA4 (red fluorescence, bottom row) was analyzed in eGFP-inducible hESCs (ZEG 3.1) before (¨COHT; middle panels) and 3 days after ( OHT; rightmost panels) induction by OHT. Cells after OHT induction are separated into GFP (right center) and red fluorescence (rightmost panels) to clarify the source of signal in identical fields. Wild-type hESCs were used as positive controls (left panels). Scale bar = 50 µm. (E): Induction of eGFP expression in EBs. eGFP-inducible hESCs (ZEG 3.1) were grown in nonadherent conditions for 3 days to form EBs, which were then cultivated in the absence (¨COHT) or presence ( OHT) of OHT for an additional 3 days. Similar induction of eGFP expression was observed when OHT treatments were performed prior to EB formation. Scale bar = 0.2 mm. (F): Fraction of cells expressing eGFP in EBs after induction by OHT. eGFP-inducible hESCs (ZEG 3.1) were grown in nonadherent conditions for 5 days to form EBs, which were then cultivated in the absence (¨COHT) or presence ( OHT) of OHT for an additional 3 days. The fraction of fluorescent cells was then determined by FACS analysis. Similar induction of eGFP expression was observed when OHT treatments were carried out prior to EB formation. (G): Induction of eGFP expression in neuroectoderm, mesendoderm, and extraembryonic tissues. eGFP-inducible hESCs (ZEG 3.1) were cultivated in nonadherent conditions in medium containing serum for 3 days. The resulting EBs were returned to adherent conditions and then grown for an additional 3 days in the absence (¨COHT) or presence ( OHT) of OHT. Expression of specific markers (red fluorescence) for mesendoderm (Sox17 and Bra), neuroectoderm (Sox2, Nestin, and Ncam), and extraembryonic tissues (GATA4 and GATA6) was analyzed by immunostaining. Cells were separated into GFP and red fluorescence to clarify the source of signal in identical fields. Nuclei are shown by Hoechst staining. Scale bar = 200 µM. Abbreviations: AP, alkaline phosphatase; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; OHT, hydroxytamoxifen.
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Figure 2. (Continued)
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Table 1. Summary of the OHT-inducible sublines generated after electroporation of the ZEG and ZNO vectors in Cre-ERT2 expressing human ESCs

Figure 3. Induction of Nodal expression limits differentiation of hESCs. (A): ¦Â-Galactosidase (¦Â-gal) expression in Nodal-inducible hESCs before and after induction by OHT. Nodal-inducible hESCs (ZNO 4.8 and ZNO 4.11) were grown in the absence (¨COHT) or presence ( OHT) of OHT, and then 2 days later, the cells were stained for ¦Â-gal expression. In the absence of OHT treatment (¨C), the majority of the cells expressed ¦Â-gal, whereas after OHT treatment ( ) ¦Â-gal expression disappeared (ZNO 4.8) or was largely reduced (ZNO 4.11). Enhanced green fluorescent protein (eGFP)-inducible hESCs (ZEG 4.5) were used as positive controls (¨C). (B): Expression of Oct-4, Nanog, eGFP, and Nodal in hESCs and their differentiated derivatives. Real-time PCR analysis was used to detect gene expression in undifferentiated hESCs grown in the absence (¨COHT) or presence ( OHT) of OHT and in the corresponding hESCs differentiated as EBs for 14 days (EBs). One eGFP-inducible line (ZEG 3.1) and two Nodal inducible lines (ZNO 4.8 and ZNO 4.11) were assessed. Relative expression reflects the CT for each samples of three informative experiments. (C): Morphology of EBs after induction of Nodal expression by OHT. Nodal-inducible hESCs (ZNO 3.2) were grown in the absence (¨COHT) or presence ( OHT) of OHT. Then, the cells were grown for 12 days in nonadherent conditions to induce EB formation. Nodal-induced EBs have outer and inner epithelial development (arrows) and thus differ from uninduced EBs. EBs developed from hESCs that constitutively express Nodal are shown for comparison (right panel). Scale bar = 100 µM. (D): Induction of Nodal expression maintains the pluripotent status of hESCs grown in chemically defined medium. Wild-type hESCs or Nodal-inducible hESCs (ZNO 4.8) were grown for 7 days without added growth factors or in the presence of Activin and FGF, Activin alone, OHT, or Activin OHT. Fluorescence-activated cell sorting was used to determine the fraction of cells expressing the pluripotency marker Tra-1¨C60. Abbreviations: FGF, fibroblast growth factor; hESC, human ESC; OHT, hydroxytamoxifen.
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Figure 3. (Continued)6 c! G9 O7 P4 y: T

Generation of hESCs Conditionally Expressing Nodal# t* g  l3 l6 V# k
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Our validation of the Cre induction approach included using it to control the expression of Nodal, a transforming growth factor ¦Â (TGF¦Â) superfamily . We grew OHT-induced ZNO-hESCs in CDM in the absence of other growth factors for 7 days and then used FACS analysis to determine the number of pluripotent cells expressing the pluripotent marker Tra-1¨C60. After OHT treatment, a majority of cells remained pluripotent, compared with only 20% in the absence of OHT (Fig. 3D). Taken together, these results exemplify the versatility of the Cre-ERT2 approach for controlling expression of a gene with key biological functions in pluripotency and differentiation of hESCs.

DISCUSSION
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The Cre-ERT2 approach has been used in a substantial number of conditional gene knockout studies in the mouse . If not managed, Cre toxicity would be a significant limitation to genetic modification of hESCs. This is especially the case for generation of reporter cell lines by "knock-in" strategies, which potentially require the excision of a selection cassette flanked by LoxP sites. Further investigation is needed to define the cellular mechanisms implicated in the cell death induced by Cre-ERT2 activity in hESCs.
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Interestingly, Nolden et al. , and thus, it will enable new experimental strategies to study mechanisms controlling cell fate specification in hESCs and in their differentiated progeny.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.2 o% @4 o4 S: {

ACKNOWLEDGMENTS
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We thank P. Chambon and D. Metzger for the Cre-ERT2 cDNA and C.G. Lobe for the ZEG vector. The SSEA-4 antibody was obtained from the Developmental Studies Hybridoma Bank. This work was supported by the Medical Research Council International Appointments Initiative and Human Frontiers Science Program grants (R.P.) and by a Diabetes U.K. Career Development Fellowship (L.V.).
      【参考文献】; N# \) z3 \. ?5 ^
8 y8 j) y/ u( u" F
9 M) o: _  I) T
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145¨C1147.

Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154¨C156.; J; |2 L# B0 N4 V, p- l

Lewandoski M. Conditional control of gene expression in the mouse. Nat Rev Genet 2001;2:743¨C755.- a+ |5 M2 L% z2 m

Mikkola HK, Orkin SH. Gene targeting and transgenic strategies for the analysis of hematopoietic development in the mouse. Methods Mol Med 2005;105:3¨C22.& s) a4 |8 d6 p' s+ N6 `, ]( ~0 b, G
% y4 f9 ^  P) n1 M9 ?: {  \8 z% S
Era T, Witte ON. Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate. Proc Natl Acad Sci U S A 2000;97:1737¨C1742.

Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372¨C376.

Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 2002;109:39¨C45.: n: r- u6 }9 i/ G
, v& `, m1 v! ^0 Z  Q- s
Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 2002;109:29¨C37.8 G" u: s* y) Q
  u& o, |; `: w4 t) S+ M
Bryja V, Pachernik J, Kubala L et al. The reverse tetracycline-controlled transactivator rtTA2s-S2 is toxic in mouse embryonic stem cells. Reprod Nutr Dev 2003;43:477¨C486.! d( n" _6 A/ ]" B( }$ R2 K
6 S5 V) e9 S* o
Masui S, Shimosato D, Toyooka Y et al. An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit. Nucleic Acids Res 2005;33:e43.
' }. E  O1 j9 B, w
Zhu Z, Zheng T, Lee CG et al. Tetracycline-controlled transcriptional regulation systems: Advances and application in transgenic animal modeling. Semin Cell Dev Biol 2002;13:121¨C128.

Indra AK, Li M, Brocard J et al. Targeted somatic mutagenesis in mouse epidermis. Horm Res 2000;54:296¨C300.
5 E/ V) D4 Y# U( E6 C, s5 p' O
Novak A, Guo C, Yang W et al. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 2000;28:147¨C155.

Vallier L, Mancip J, Markossian S et al. An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives. Proc Natl Acad Sci U S A 2001;98:2467¨C2472.  b4 f# a; r5 D: |8 Q8 z
5 l( V% f! }3 \6 v1 G9 E
Vallier L, Reynolds D, Pedersen RA. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol 2004;275:403¨C421.  H9 u/ J) P' u

Vallier L, Rugg-Gunn PJ, Bouhon IA et al. Enhancing and diminishing gene function in human embryonic stem cells. STEM CELLS 2004;22:2¨C11.

Vallier L, Alexander M, Pedersen RA. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 2005;118:4495¨C4509.) R* D' g9 g: n. Y- Y
7 M! L  s% s% ~7 X" D' ?( O1 j& ^
Baba Y, Nakano M, Yamada Y et al. Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells. Microbiol Immunol 2005;49:559¨C570.
8 k8 t2 E* D) Q, D5 K5 v
Loonstra A, Vooijs M, Beverloo HB et al. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A 2001;98:9209¨C9214.! n! ^2 f# s' v& {  Q! d
/ \+ _8 ?! |  J, W/ N
Silver DP, Livingston DM. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol Cell 2001;8:233¨C243.# J* \2 V9 f* h$ r! F: {5 m
  _" s* J( _0 `1 k. i
Sauer B. Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance. J Mol Biol 1992;223:911¨C928., V3 y2 y3 U0 |& `& z

Thyagarajan B, Guimaraes MJ, Groth AC et al. Mammalian genomes contain active recombinase recognition sites. Gene 2000;244:47¨C54.
2 M* Z% V  k6 w2 E
Sauer B. Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome. Nucleic Acids Res 1996;24:4608¨C4613.1 v. k# e6 G# K/ L, r! @( A, O9 X
) E+ D0 v  u5 R9 V% n
Schmidt EE, Taylor DS, Prigge JR et al. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci U S A 2000;97:13702¨C13707.

Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol 2003;19:589¨C621.
4 `& K1 J( e$ N; t, _, b% K5 k
Feil R, Brocard J, Mascrez B et al. Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A 1996;93:10887¨C10890.; p3 ?8 P  N6 s7 U' i# A6 E2 w
! q9 @: G' ~$ v' Y: }1 E) \4 ~( s
Brocard J, Warot X, Wendling O et al. Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci U S A 1997;94:14559¨C14563.

Weber P, Metzger D, Chambon P. Temporally controlled targeted somatic mutagenesis in the mouse brain. Eur J Neurosci 2001;14:1777¨C1783., n0 _2 |% r$ W$ U" E
& q, X2 I5 }0 |
el Marjou F, Janssen KP, Chang BH et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 2004;39:186¨C193.- R( c. j) B$ |; f+ N2 I6 r
. {; R1 W: g& D* Q5 h- B' g
Schuler M, Ali F, Metzger E et al. Temporally controlled targeted somatic mutagenesis in skeletal muscles of the mouse. Genesis 2005;41:165¨C170.0 ~' {$ v2 @" j& [/ B/ T' g
, N$ [' v$ t& i6 F1 F5 ~
Nolden L, Edenhofer F, Haupt S et al. Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase. Nat Methods 2006;3:461¨C467.. P  a1 w* {( x$ D" b

Dor Y, Brown J, Martinez OI et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004;429:41¨C46.