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Enhancing and Diminishing Gene Function in Human Embryonic Stem Cells

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发表于 2009-3-5 10:22 |显示全部帖子
a Department of Surgery, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom;
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b School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom
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  v* n+ Z7 h! fKey Words. Human embryonic stem cells ? Fluorescent reporter genes ? Green fluorescent protein ? Red fluorescent protein ? Transfection ? Small interfering RNA ? Gain-of-function ? Loss-of-function ? Stem cell therapy ? Regenerative medicine
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Ludovic Vallier, Ph.D., Box 202, Level E9, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, United Kingdom. Telephone: 44-1223-762025; Fax: 44-1223-410772; e-mail: lv225@cam.ac.uk
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ABSTRACT1 u+ s/ d6 s% P; t( o
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The unique value of human embryonic stem (hES) cells for regenerative medicine lies in their capacity to differentiate into all body tissues . Mouse ES cells expressing lineage-specific transcription factors are capable of providing clinical benefits for immunodeficiency or Parkinson’s disease when their in vitro differentiated derivatives are transplanted into recipient animal models . Thus, robust technologies for enhancing and diminishing gene function in hES cells can expedite their controlled in vitro differentiation into clinically useful cell types. This may involve, for example, achieving the expression of inductive genes, inhibiting mechanisms leading to competing pathways, and supplying reporter genes to indicate the status of cell differentiation. We used lipofection to generate hES cell lines expressing naturally fluorescent green or red proteins without changing their characteristics, specifically their capacity to differentiate into mesoderm, endoderm, and ectoderm cell types. We used these cell lines to examine the utility of small interfering (si)RNAs  as a means of diminishing hES cell gene function. We found that siRNAs were an effective and specific means of knocking down gene expression in hES cells whether administered transiently by lipofection or permanently by expression of hairpin-loop siRNA.3 d6 W: j3 h$ n/ k, I: D- R0 Q
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MATERIALS AND METHODS
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  ^7 S5 Y' \+ s2 a+ l/ PFluorescent reporter genes provide for a quantitative and qualitative estimation of specific gene expression directly in living cells, using simple assays such as fluorescence microscopy or FACS, which are capable of single-cell resolution . However, the expression of fluorescent reporter proteins at a sufficient level to be readily detected can be toxic for some cell types .) w5 |6 h- s, y; V
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We tested five different fluorescent proteins (destabilized d2GFP , eGFP , TauGFP , hrGFP , and DsRed2 ) in mouse ES cells to identify those with maximal brightness and minimal toxicity. Each fluorescent reporter gene was subcloned downstream of the CAGG promoter into the pTp6 expression vector  (Fig. 1A) in order to obtain equivalent levels of expression (data not shown). The CAGG promoter confers strong and ubiquitous expression in mouse (m)ES cells and in mice derived from them . Moreover, by linking the fluorescent reporter gene to the puromycin resistance gene through an internal ribosome entry site (IRES) , strong selective pressure could be applied on transgene expression during isolation and subsequent passaging of transfected colonies. Of the fluorescent reporter genes tested, hrGFP was the brightest and one of the least toxic for mES cells (Fig. 1B, 1C). Furthermore, silencing of this gene was observed only at low levels in mES cells (Fig. 1D), and the green protein was resistant to paraformaldehyde fixation, allowing its use for immunochemistry (data not shown). As a complementary fluorescent reporter gene, DsRed2 was chosen for its brightness and protein stability, although it showed a tendency towards silencing when mES cells were passaged repeatedly without drug selection (Fig. 1D). While recent results suggest lentiviral vectors have a high transfection efficiency when used in hES cells , they have the disadvantage of being complex to generate and accommodate a limited size of transgene. However, there is no clear consensus on relative efficiencies of the more conventional approaches to transfection of hES cells . We thus examined the efficiency of several transfection reagents in preliminary experiments (data not shown). On this basis, we then compared Lipofectamine 2000 and Exgen 500  for stable transgene expression in hES cells using hrGFP and DsRed2 (Fig 2A, 2C). Colonies generated using the hrGFP vector with Lipofectamine transfection (n = 40), as well as those generated using Exgen 500 (n = 10) retained green fluorescence after five passages without puromycin selection. However, only 6 of 48 DsRed2-expressing clones retained red fluorescence after five passages without selection. This reduction in fluorescence was lessened and sometimes reversed by resuming puromycin selection. Evidently, DsRed2 is sensitive to epigenetic silencing in hES cells, confirming the results obtained with mouse ES cells (Fig. 1D), thus requiring their continuous maintenance under drug selection. After 12 passages (2 months of culture), FACS analysis revealed that 99% of cells were fluorescent in several green- and red-fluorescing hES cell lines selected for optimal expression by fluorescent microscopy (Fig. 2B).
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+ {! n( i8 N% {2 C. ]  o% RFigure 1. Toxicity and stability of different fluorescent reporter genes in mouse (m) ES cells. A) Map of the pTP6 vector. pTP6 vector was developed by Pratt et al. . It contains the CAGG promoter followed by the TauGFP gene and an IRES puromycin-resistance gene. All the GFP-pTP6 vectors were constructed replacing the TauGFP by the corresponding fluorescent reporter gene. Thus, the expression of each fluorescent gene was under the control of the CAGG promoter. B) Fraction of fluorescent cells after transitory electroporation. The R1 and CGR8 mES cell lines were transitorily electroporated with pTP6 vectors expressing DsRed2, d2GFP, eGFP, TauGFP, or hrGFP.
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4 x2 R7 U! f# v# W$ ]+ R! I6 uThe electroporation conditions used allowed transfection of a large fraction of cells with a high quantity of DNA, resulting in strong expression. Consequently, if the fluorescent protein were toxic, the electroporated cells would be expected to die. A control vector was co-electroporated with each pTP6 vector to assess variations due to electroporation efficiency. hrGFP gave better results than DsRed2 and d2GFP (p = 0.02, 10 degrees of freedom ). However, the fraction of fluorescent cells obtained with TauGFP, eGFP, and hrGFP was not significantly different from each other (p = 0.6, 10 d.f.). C) Stable expression. The R1 and CGR8 mES cell lines were stably electroporated with pTp6 vectors encoding each fluorescent protein. After 10 days of puromycin selection, the number of clones obtained with each reporter gene was counted. Because use of an IRES vector construction linked the level of fluorescence reporter gene expression to that of puromycin resistance gene expression, all the drug-resistant clones were fluorescent. Consequently, if the fluorescent protein were toxic, the clone would be expected to die during selection, so that fluorescent reporter genes with high clone yields can be regarded as low in toxicity and, conversely, those with low clone yields as toxic or detrimental. TauGFP gave the lowest number of clones (p = 0.03, 4 d.f.). The number of clones generated with the other fluorescent genes was not significantly different from each (p = 0.4, 4 d.f.). To assay the brightness of each fluorescent protein, we selected clearly positive clones using fluorescence microscopy and then evaluated their brightness using FACS (data not shown). D) Sensitivity to silencing. The brightest fluorescent clones for each reporter gene were selected by fluorescence microscopy and then grown for 7 weeks without puromycin selection. The fraction of fluorescent cells was analyzed by FACS to determine whether there was an increase in the number of the negative cells over time (defined as silencing). DsRed2 underwent silencing (>30% of cells), whereas other fluorescent genes did not to a substantial degree (
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Figure 2. Generation of hES cell lines stably expressing red and green fluorescent reporter genes. A) Number of transgenic colonies generated using Lipofectamine 2000 and Exgen 500. H9 hES cells were transfected with DsRed2-pTp6 or hrGFP-pTp6 as described in experimental protocols. The same number and condition of hES cells were used in each case. No significant differences were observed when transfecting linearized or circular DNA. The highest number of stably expressing cell lines was generated using Lipofectamine 2000 (p = 0.05, 6 d.f.). B) FACS analysis of red and green hES cell lines that were selected by fluorescent microscopy as clearly expressing fluorescent protein. C) hES cell colonies expressing red and green fluorescent proteins (upper left and upper right, GFP fluorescence; corresponding phase contrast images are shown in lower left and right panel, respectively). D) Differentiated hES cells expressing red and green fluorescent proteins. Embryoid bodies were generated by growing hES cells in nonadherent conditions for 5 days, after which they were plated for 2 weeks on 12-well gelatin-coated plates. E) ? tubulin expression in differentiated red fluorescent hES cells. Embryoid bodies were generated by growing hES cells in nonadherent conditions for 5 days, after which they were plated for 2 weeks on 12-well gelatin coated plates. Neurosphere-like structures appeared spontaneously 1 week after plating. These structures expressed the ? tubulin protein (green fluorescence), which is a specific marker for neurons.
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After hES cell differentiation in vitro, fluorescent protein expression was strongly detected in beating structures presumably containing cardiomyocyte (mesoderm)-like cells. Green or red fluorescent neuron (ectoderm)-like cells were also observed, and these cells expressed the specific neuronal marker ? tubulin (Fig. 2D, 2E). Also, RT-PCR was used to detect the expression of  fetoprotein, albumin, and somatostatin, which are specific markers of endoderm  (data not shown). These results show that green and red fluorescent hES cells can be differentiated into all three germ layers. However, further studies will be needed to assess the functionality of differentiated cells derived from fluorescent hES cells. Finally, the pluripotent stem cell markers, Oct-4 and FGF-4, as well as the embryonic stem cell surface markers, SSEA-4, Tra-160, and Tra-181, were all expressed in fluorescent reporter transgene-expressing hES cells, whereas the differentiation marker SSEA-1 remained absent (data not shown). Furthermore, the karyotype and morphology of these cells were normal (data not shown), so despite the transfection and the selection process, the red- and green-fluorescing hES cells retained molecular markers of their undifferentiated state as well as the ability to differentiate into derivatives of each of the three germ layers. These findings provide additional compelling evidence that such genetic alteration of hES cells is compatible with maintenance of their pluripotency . This expands the use of fluorescent reporter-expressing hES cell lines as powerful tools for cell tracing during tissue differentiation in in vitro and in animal models.
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; |0 i2 j5 p6 v' k. O$ VThe principal approach that has been used to study gene function in mammals is gene targeting by homologous recombination in mES cells . Such an approach recently has been used with hES cells to generate a mutation in a single allele of the HPRT and Oct-4 genes . However, generating a homozygous null mutation would require targeting of the second allele in the case of autosomal genes, or might be accomplished less reproducibly by gene conversion . To circumvent this obstacle to perturbing gene function in hES cells, we evaluated the use of short-interfering double-stranded RNA, which has been shown to be efficient in mouse ES cells .( H/ X3 v7 ^+ f" y3 ~: l
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We first sought to define the efficacy of siRNA in hES cells by targeting the hrGFP and DsRed2 genes in the fluorescent cell lines generated above using transitory transfection of siRNAs. Fluorescent reporter genes provide distinct advantages as siRNA targets, in that they are capable of single cell assessment, can sustain multiple observations in living cells without perturbing viability and development, and they encode readily detectable proteins, whose absence does not alter cellular phenotype or provide a proliferative disadvantage. We designed three siRNA oligonucleotides targeted against the mRNA of each gene and transfected them into stably expressing green or red hES cell lines using Oligofectamine. After 48 hours, we observed the disappearance of fluorescence in a fraction (5%–10%) of the cells. To increase this effect, cells were retransfected 24 hours after the initial transfection. The siRNA effect was analyzed 48 hours later by counting the number of colonies containing negative cells using fluorescence microscopy (data not shown), by counting the negative cells using FACS (Fig. 3A), or by direct measurement of fluorescent gene expression using real time RT-PCR (Fig. 3C). Two of three anti-green siRNAs (14 and 22) reduced the expression of the green protein in 10% and 20% of the cells, respectively (Fig. 3A) (t-test, p = 0.007), and negative cells were detected in all the colonies treated with them (Fig. 3B).7 l2 H/ ^: Q  l! ?: z
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Figure 3. Effect of siRNAs on red and green fluorescent protein expression in hES cells. A) FACS analysis of the siRNA effect on hrGFP expression. Three different siRNAs (anti-green 6, anti-green 14, and anti-green 22) designed against hrGFP mRNA were transfected twice into hES cells expressing the hrGFP fluorescent reporter gene. Two days after the second transfection, fluorescent cells were counted using FACS. No siRNA effect was detected 24 hours before or 24 hours after this time. The transfection procedure itself did not affect hrGFP expression, since no decrease in fluorescence was observed when only water was transfected (p = 0.7, 2 d.f.). The transfection of anti-red siRNA did not diminish hrGFP expression, whereas it did diminish DsRed2 expression (p = 0.007, 2 d.f.). B) Diminished gene expression induced by siRNA in fluorescent hES cells. Red and green fluorescent hES colonies containing negative cells induced by transfecting their respective siRNAs. C) Real-time RT-PCR analysis of the siRNA anti-green 22 effect on hrGFP expression of green fluorescent hES cells. Three days after the first transfection, normalized GFP/PBGD mRNA levels were measured using RT-PCR. Results are shown in comparison to untransfected control cells (100%). A significant decrease (40%) of the hrGFP RNA was observed with anti-green siRNA (p = 0.006, 4 d.f.) but not in response to siRNA directed against DsRed2 or to water (p = 0.1, 4 d.f.). D) Real-time RT-PCR analysis of the siRNA effect on endogenous gene expression. Green fluorescent hES cells were transfected as described in 4C. Two days after the second transfection, normalized ?2M/PBGD mRNA levels were measured by real-time PCR. Results are shown in comparison to untransfected control cells (100%). siRNA transfection did not affect the expression of the ?2M gene (p = 0.8, 4 d.f.). E) Expression of pluripotent markers in siRNA transfected hES cells. Two days after the second transfection, hES cells were immunostained for the SSEA-1, SSEA-4, and TRA-1-60 surface antigens. The SSEA-1 antigen is a marker of differentiated cells, while SSEA-4 and TRA-1-60 are specific markers of undifferentiated hES cells. For each antigen, the first row  shows untransfected hES cell colonies and the siRNA row shows red fluorescent hES cell colonies transfected with anti-red siRNA. The green fluorescence corresponds to the surface-antigen staining. No differences were observed between untransfected and transfected cells, suggesting that siRNA transfection did not change the pluripotent state of hES cells. Moreover, the normal expression of these markers shows that the siRNA effect is specific for the targeted gene and did not interfere with gene expression in general.& x3 X- D6 k( k/ ^

) R6 V* i6 O0 [# y& o4 JWe observed similar effects with one of three siRNA against DsRed2 mRNA in red hES cells, where a majority of colonies contained negative cells (Fig. 3B and data not shown). The effect of the anti-red siRNA was more difficult to assay by FACS because of the stability of the red protein (data not shown). At the RNA level, anti-green siRNA 22 induced a 40% decrease in the green fluorescent gene transcript (Fig. 3C), suggesting that siRNA had a greater impact at the RNA level than at the protein level (Fig. 3A and 3C). Thus, transient transfection of siRNA led to a decrease of gene products in a substantial fraction of cells; however, the knock down apparently did not reach a null level.$ X1 j! \( N$ g

, q( {9 c( R  U2 K! c" sThe siRNA-induced knock down of gene function was specific to the targeted gene. The transfection of anti-red siRNA into green hES cells or anti-green siRNA into red hES cells had no effect, respectively, on hrGFP or DsRed2 expression as compared with the controls. Nonspecific effects were similarly absent when we examined the expression of endogenous genes using real-time PCR (Fig 3D). Likewise, siRNA transfection into hES cells did not alter expression of pluripotent markers such as Oct-4 (data not shown), SSEA-4, or Tra-1-60 (Fig. 3E). Therefore, siRNA does not appear to activate the interferon response or the PKR kinase pathway, which would lead to a generalized suppression of transcription, as with long double-stranded RNAs, nor does it affect the expression of markers indicative of the pluripotent state. Therefore, these findings provided proof of principle that siRNA knocks down gene expression in hES cells, although the fluorescent reporter genes were challenging targets for detecting such effects. First, the sensitivity of detecting the fluorescent proteins made it difficult to assess the degree of knock down at the protein level. This, together with the short-term, heterogeneous nature of transitory transfection, meant that we could not isolate a homogenous population of affected cells on which to accurately assess the extent of knock down at the mRNA level. Thus, the transient transfection data did not allow us to distinguish between an intermediate knock down efficiency at the mRNA level in most of the cells versus a high-efficiency knock down in a fraction of the cells.
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+ ~7 d- F* A, {) TAccordingly, we sought to establish hES cell sublines stably expressing hairpin-loop siRNA as a means of obtaining homogenous populations of cells. The characteristics of a desirable protein target for such an analysis would include being selectable and being nonessential for viability, pluripotency, or differentiation. On this basis, we targeted the endogenous HPRT gene, which encodes an enzyme involved in purine metabolism . Because 6-TG is metabolized to a toxic compound by HPRT, it can be used to select for and identify drug-resistant cells that are deficient in HPRT. Using this approach, we generated five hES sublines that were 6-TG resistant among 10 sublines generated by cotransfection and selection for puromycin resistance, and another five sublines that were obtained by direct 6-TG selection (Table 1; Fig. 4A). Interestingly, none of the 10 hES sublines were sensitive to medium containing hypoxanthine, aminopterin, and thymidine (HAT), which blocks DNA synthesis in cells that are HPRT deficient, suggesting that while HPRT levels are diminished sufficiently to provide 6-TG resistance, they are not reduced to a null level. Consistent with this hypothesis, the levels of HPRT mRNA (analyzed by semiquantitative RT-PCR) were diminished to the borderline of detectability in siRNA-expressing hES cells (Fig. 4B)., e: ]9 |8 j; m3 ~( v7 ]7 o* j
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Table 1. Summary of siRNA-expressing hES cell sublines generated by stable transfection of the pSuper-HPRT vector
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Figure 4. Knock down of HPRT gene in undifferentiated hES cells stably transfected with pSuper-HPRT hairpin loop vector. A) 6-TG resistance of hES cells stably transfected with pSuper-HPRT vector. Nontransfected hES cells (control) and stably transfected hES cells (siRNA HPRT) were grown during 7 days in normal medium (knock out serum replacement ) or media containing either HAT (1x) or 6-TG (2 μg/ml). The surviving colonies were stained using crystal violet. No hES cell colony was detected in controls after 7 days of 6-TG selection (upper right well), while a majority of siRNA-expressing hES cell colonies survived (bottom right well). Both kinds of cells survived HAT selection, showing that HPRT is not knocked down to a null level in transfected hES cells. B) Analysis of HPRT expression in hES cells stably transfected with pSuper-HPRT vector. HPRT expression was analyzed using semiquantitative RT-PCR based on a time course of PCR cycle allowing comparison within the log phase. HPRT expression was almost undetectable in transfected hES cells resistant to 6-TG (HPRT1 and 6TG2), while it was readily detectable in nontransfected cells (control) or in transfected hES cells that were sensitive to 6-TG (HPRT2). FGF-4, a marker of pluripotency, and ?2M were both expressed at the same level in transfected hES cells (HPRT1, HPRT2, 6TG2) and in nontransfected cells (control), showing specificity of the siRNA effect.7 d. Z3 S% p3 D& g0 T

/ l* D; K1 V/ m9 g- R8 oUnder the same assay conditions, HPRT expression was readily detectable in wild-type hES cells and in an hES cell subline that was puromycin resistant but 6-TG sensitive (HPRT2). We also assayed the effect of siRNA expression after hES-derived cells had differentiated into embryoid bodies for 6 days, were plated in normal medium for 5 more days, and, finally, were subjected to 6-TG selection for an additional 12 days. At the end of this selection regimen, few wild-type cells survived the 6-TG selection, whereas a majority of siRNA-expressing cells were still alive (Fig. 5A). Therefore, stable expression of siRNA is also effective in differentiated cells derived from hES cells. Our analysis of HPRT gene expression in differentiated cells using semiquantitative RT-PCR revealed almost undetectable mRNA levels, whereas wild-type hES cells expressed HPRT at high levels (Fig. 5B).8 Y: _, G; g! f

$ g; b' r! K9 @; RFigure 5. HPRT knock down in differentiated derivatives of hES cells stably transfected with the pSuper-HPRT vector. A) 6-TG resistance of differentiated derivatives of transfected hES cells. Embryoid bodies (EBs) were generated from stably transfected hES cells by growing them in nonadherent conditions for 5 days. Then EBs were plated on gelatin-coated plates containing normal medium for 5 more days and, finally, were subjected to 6-TG (2 μg/ml) or HAT (1x) selection for an additional 12 days. The same number of EBs was plated for each well. After 2 weeks, surviving cells were stained using crystal violet. Nontransfected cells (control) did not survive in 6-TG-containing medium (top right well), while a majority of siRNA-expressing cells (siRNA HPRT) survived in 6-TG medium (bottom right well). Both kinds of cells survived in HAT medium (middle wells). (B) Analysis of HPRT expression in differentiated derivatives of hES cells stably transfected with the pSuper-HPRT vector. HPRT expression was analyzed using semiquantitative RT-PCR based on a time course of PCR cycle allowing comparison within the log phase. HPRT expression was barely detectable in differentiated derivatives of transfected hES cells resistant to 6-TG (HPRT1, HPRT4, and 6TG2), while it was readily detectable in nontransfected cells (Control). Cyclin D1 and ?2M were expressed at the same level in differentiated derivatives of transfected hES cells (HPRT1 and 6TG2) and in nontransfected cells (control) showing specificity of the siRNA effect.1 ]( T) u) T  p* Q8 w. Y
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Housekeeping genes such as ?2M and Cyclin D1 were expressed at wild-type levels in both control cells and in siRNA-expressing cells, showing the specificity of the siRNA effect on the HPRT target gene. Taken together, these results show that stable expression of siRNA knocks down both transgenic and endogenous genes expressed in hES cells and in their differentiated derivatives. While the knock down does not reach a null level, the resulting hypomorphic phenotypes can nevertheless provide both developmental and clinical models for understanding human functional genomics. For example, mouse mutants with various expression levels of vascular endothelial growth factor family genes have provided insights that could not be gained from the null mutation, which is lethal just after gastrulation stages . Similarly, a hypomorphic condition for HPRT may provide models not only for Lesch-Nyhan syndrome, which is associated with null or very low HPRT levels, but also for Kelley-Seegmiller syndrome and gout, which are associated with intermediate levels . In these cases, there are no animal models because HPRT-deficient mice are healthy. Thus, siRNA approaches involving hES cells and their differentiated derivatives can provide a key resource for human functional genomics, complementing gene targeting by homologous recombination that results in complete ablation of the targeted gene .
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0 V1 W' W) L' g: B1 P. xThe potential for clinical application of hES cells is encouraging; however, this potential is still undergoing biological and technical evaluation. Beyond the obviously important task of controlling stem cell differentiation, realizing the clinical goal of cell-based transplantation therapies will require a means of matching donor tissues to the host immune system  and will also necessitate the derivation of new hES cell lines without using animal cells as feeders . Each of these major tasks will benefit from robust approaches for regulating the expression of specific exogenous and endogenous genes. For example, use of siRNA could reveal the function of genes involved in the maintenance of pluripotency and thus lead to improvements in the efficiency of hES cell derivation, which would be necessary for generation of an hES cell bank. The results described here provide evidence that hES cells can undergo alteration in the expression of specific genes without effects on their pluripotency, a fundamental developmental characteristic of hES cells. Finally, because hES cells are capable of undergoing some of the differentiative events characteristic of early human development, these approaches for monitoring and modifying hES cell gene expression provide a useful model for human functional genomics. This in turn can reveal the molecular pathways of differentiation into the diversity of cells and tissues that ES cells are capable of forming.) X, \! ^! q0 ?' X% e- x

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发表于 2015-6-4 22:54 |显示全部帖子
淋巴细胞

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给我一个女人,我可以创造一个民族;给我一瓶酒,我可以带领他们征服全世界 。。。。。。。。。  

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发表于 2015-6-27 13:43 |显示全部帖子
干细胞之家微信公众号
我等你哟!  

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先看看怎么样!  

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天啊. 很好的资源

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我回不回呢 考虑再三 还是不回了吧 ^_^  

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我卷了~~~~~~~  

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真是有你的!  

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今天临床的资料更新很多呀
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