Screening for Genes Essential for Mouse Embryonic Stem Cell Self-Renewal Using a - 《stemcells》干细胞学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 21 下一页

返回列表

查看: 336864\|回复: 204	go Screening for Genes Essential for Mouse Embryonic Stem Cell Self-Renewal Using a [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-4 23:58 |只看该作者 |倒序浏览 |打印

作者：Jun-Zheng Zhang, Wei Gao, Hong-Bo Yang, Bo Zhang, Zuo-Yan Zhu, You-Fang Xue作者单位：Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing, China

   ( U9 }- n* I! T! v7 W5 f8 O


         5 ]8 V. R( H" B* o

         9 D( O4 Q- r* A2 j' K/ n. Y6 j



      - F; ]0 i: f' u( @( `& u

      【摘要】3 d: @+ [. c4 r
   The pluripotency of mouse embryonic stem (ES) cells is maintained by self-renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct-4 were monitored 48 hours later by real time RT-PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct-4 expression to less than 50% of the normal level and 7 of them upregulated Oct-4 expression to more than 150% of the normal level. To investigate their long-term effect on self-renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418-resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex-1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self-renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self-renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.
      【关键词】 Self-renewal RNA interference library Mouse embryonic stem cells Oct- Zfp/Rex-( u1 j7 v* m; x/ C
   INTRODUCTION

Since the initial derivation from preimplantation mouse embryos in the early 1980s, mouse embryonic stem (ES) cells have been shown to have nearly unlimited developmental potential both in vitro and in vivo, which was defined as pluripotency (reviewed in . When raised to more than 150% of wild-type levels, the increase in Oct-4 expression causes differentiation into extraembryonic endoderm and mesoderm; whereas a 50% loss of Oct-4 expression induces the formation of trophectoderm. Thus Oct-4 expression level is a critical marker for ES cells' self-renewal capability.: ~/ o. F. F; D2 A# Q$ v8 j1 @
6 A) L3 N6 j( o4 i" [! C; G$ ^
Several techniques, including serial analysis of gene expression, representational difference analysis, cDNA microarray analyses, and oligonucleotide microarray analyses, were used to identify genes potentially involved with ES cell self-renewal (reviewed in , suggesting potential limitation of this approach.

We developed a functional screen for genes essential for ES cell self-renewal based on RNA interference (RNAi) technology. RNAi is a double-stranded RNA (dsRNA)-mediated gene knock-down technology first discovered in Caenorhabditis elegans , suggesting the possibility of dissecting self-renewal mechanism in ES cells with RNAi technology. Here we report the establishment of an RNAi library constructed from subtracted mouse ES cell cDNAs and the use of the library to screen for genes essential for ES cell self-renewal. After two rounds of screening, in total eight genes were identified, including some previously predicted genes (such as Zfp42, Dppa5 et al.) as well as some unreported genes. Furthermore, we showed that one candidate gene, Zfp42, played an important role in ES cell self-renewal. This is the first time that RNAi technology was used to systemically study the mechanism of ES cell self-renewal, and our results proved that the RNAi library we constructed is a feasible and efficient tool to screen for genes involved in this important progress. Further studies about the genes we identified will help us understand the self-renewal property of ES cells.
) u7 Y( D8 s" ~
MATERIALS AND METHODS1 F- X: D5 K$ C1 g4 b, \- F

Plasmids Construction

The RNAi plasmid pDC-EGFP (Fig. 1A) was constructed as follows. The cytomegalovirus (CMV) promoter amplified by polymerase chain reaction (PCR) (primer CMV-S and CMV-A) from plasmid pEGFP-C1 (Clontech, Palo Alto, CA, http://www.clontech.com) was inserted as an inverted repeat into the ApaI and KpnI sites of pEGFP-C1. The SV40 polyadenylation signal poly(A) fragment isolated from pEGFP-C1 plasmid with SmaI and MluI enzymes were blunt-ended with T4 DNA polymerase (Takara) and then ligated into the AseI site near the original CMV promoter of the double-promoter construct generated above. The enhanced green fluorescent protein (EGFP) fragment can be released by double digestion of NheI and HindIII to get pDC backbones. The pDC-Oct4 plasmid was constructed by replacing EGFP (NheI and BglII) with a 485-base pair (bp) Oct-4 cDNA fragment amplified from mouse ES cDNAs (primer Oct-1S and Oct-1A). U6shZfp42 and U6shZfp42GFP plasmids were constructed as described before (, supplemental methods). The CMV-EGFP-Zfp42 plasmid was constructed by inserting Zfp42 cDNA into BglII/EcoRI sites of pEGFP-C1 plasmid.
5 n( `' F+ y& }0 T$ T
Figure 1. Construction of plasmid pDC-EGFP and RNA interference (RNAi) library. (A): The pDC plasmid contains two opposing CMV promoters and poly(A) signals downstream from each of the promoters; thus any cDNA fragment inserted between them can be transcribed from both directions and form double-stranded RNA to induce RNAi effect. (B): The RNAi library was constructed by ligating subtracted ES cDNAs into the pDC plasmid backbone. Subtracted ES cDNAs contained adaptor-1 and adaptor-2R at each end, respectively, which were used as PCR primers binding sites to confirm insertion of cDNA fragments. RNAi plasmids within inserts were extracted, and subsequent screening in M13 ES cells was performed. Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; PCR, polymerase chain reaction.* }  X0 h6 K& A8 y
. H: H, L  b- @. u; I, p
ES Cell Culture and cDNA Subtraction

The ES cell line MESPU13 (M13) was established , supplemental methods). Total RNAs were extracted from MESPU13 ES cells and mouse embryonic fibroblast (MEF) cells by TRIzol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and mRNAs were isolated using the PolyA Tract mRNA Isolation System III Kit (Promega, Madison, WI, http://www.promega.com). cDNA synthesis and subtraction were performed with the PCR-Select cDNA subtraction kit (Clontech) according to the manufacturer's instructions. Twenty-five cycles of primary PCR and 12 cycles of secondary PCR were performed.
9 P" y1 M2 H, y3 K/ b1 |
RNAi Library Construction
+ ^% L: _# ^3 J6 R
Backbones for the RNAi library were obtained from NheI and HindIII double-digested pDC-EGFP plasmids. Subtracted cDNA fragments were blunt-ended by T4 DNA polymerase and ligated into backbones. Part of the library was then electroporated into DH5 Escherichia coli cells (Takara, Otsu, Japan, http://www.takara.co.jp) according to the manufacturer's instructions. After incubating overnight at 37¡ãC on LB plates containing 50 µg/ml kanamycin, single colonies were picked and inoculated into 5 ml of LB containing 50 µg/ml kanamycin. After shaking at 37¡ãC overnight at the speed of 260 rpm, 1 µl of bacteria was used as the PCR template to detect the insertion of cDNA fragments. PCR primers are the same as the nested PCR primers (PCR-1 and PCR-2R) used in the secondary PCR amplification of cDNA subtraction. Plasmids with inserts were then extracted from 5 ml of LB using the SV Minipreps DNA Purification System (Promega) (Fig. 1B).7 C5 n/ d7 G) M2 r* H- n0 p

ES Cell Transfection7 r9 Z' o! S; `+ N& @7 X

ES cells were transfected by either Lipofectamine 2000 (Invitrogen) or electroporation. One µg of plasmid and 3 µl of Lipofectamine were used for each well of a 24-well plate according to the manufacturer's instructions. When G418 selection was arranged, ES cells were trypsinized and incubated with DNA-Lipofectamine mixture for 10 minutes before being removed to gelatin culture plates. Ten µg of plasmid DNA was normally electroporated into one 3.5-cm dish of ES cells under the condition of 250 V, 500 µF using the Bio-Rad GenePulser System (Bio-Rad, Hercules, CA, http://www.bio-rad.com).8 q2 V  q' {, N3 j
: o1 `) }4 y7 S, z
Real Time Reverse Transcription-Polymerase Chain Reaction. {$ a! V" E' J( _' @1 e; v

ES cells were harvested 48 hours after transfection, and RNAs were extracted by TRIzol. The reverse transcription reaction was performed using AMLV reverse transcriptase (Promega). Quantitative real time reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the DNA Engine Opticon 2 system (Bio-Rad). Oct-4 expression level was detected (primer Oct3-S and Oct3-A), and ß-actin was measured as internal control (primer Act3-A and Act3-S).- d; X* ]0 f. m6 v2 Q- T  L
8 K) Y& T' _& ~$ q0 G3 H3 t
Alkaline Phosphatase Staining

ES cells were fixed in 4% paraformaldehyde (Sigma, St. Louis, http://www.sigma-aldrich.com) in phosphate-buffered saline (PBS) for 5 minutes at room temperature and rinsed two times by PBS. After rinsing again with 25 mM Tris-Cl (pH 9.0), freshly prepared alkaline phosphatase (AP) staining solution was added, and cells were stained at room temperature for 15¨C30 minutes. AP staining solution contained 0.4 mg/ml -naphthyl phosphate (Sigma), 1 mg/ml Fast Red TR (Sigma), and 8 mM MgCl2 (Sigma) in 25 mM Tris-Cl (pH 9.0).% Q( h' X7 [) f
  L$ R$ r9 c" y2 q5 O
RESULTS
# ~+ q, g; }0 @4 a1 F/ q; f& U
The pDC RNAi Plasmids Are Highly Effective in Undifferentiated ES Cells
5 _6 A* B( M2 V" V2 c# `
The RNAi effect of the pDC plasmids was validated by their ability to repress reporter gene and endogenous gene expression. When a transgenic ES cell line M13G2A stably expressing EGFP (Fig. 2A, 2B) was electroporated with 10 µg of pDC-EGFP plasmid, EGFP expression was obviously knocked down in most colonies, and even totally lost in some colonies (Fig. 2C, 2D). When 5 µg of pDC-Oct4 plasmid was transfected into M13 ES cells by Lipofectamine 2000, quantitative real time RT-PCR results showed that 48 hours after transfection, pDC-Oct4 could repress Oct-4 mRNA level to less than 50% of the control group (Fig. 2E). This suppression efficiency was comparable with the widely used small hairpin (sh)RNAi plasmid U6shOct4GFP (Fig. 2E), indicating the feasibility of using pDC plasmids to knock down endogenous genes.! {: F3 ~1 f0 L! w. W% c

Figure 2. RNA interference effect of pDC-EGFP and pDC-Oct4 plasmids. Phase contrast and EGFP fluorescent image of normal M13GA ES cells (A, B) and pDC-EGFP plasmid electroporated M13G2A cells (C, D) are shown. Original magnification is x40. (E): Quantitative real time reverse transcription-polymerase chain reaction (RT-PCR) results of Oct-4 mRNA levels of ES cells transfected by pDC-Oct4 (black column), pDC-EGFP (white column), and U6shOct4GFP (gray column) plasmids. Oct-4 expression level of pDC-EGFP transfected ES cells was taken as the normal value (defined as 1.0). Two groups of transfection were done independently, real time RT-PCR experiments were repeated three times for each transfection, and standard deviations were calculated. ß-Actin was used as an internal control. Abbreviations: EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein.1 U% a/ j# C2 Z9 P, Y+ L: ]4 e
- J$ A3 }5 [: Z2 r% _! }
Construction of a Subtractive RNAi Library and Screening for Self-Renewal Related Genes

M13 ES cell cDNAs were subtracted with MEF cells cDNAs; subtraction efficiency was verified by PCR tests (supplemental Fig. 1A). Subtracted cDNAs also proved to yield good diversity (supplemental Fig. 1B). The subtracted cDNA fragments were cloned into the pDC plasmid to produce an initial RNAi library. Nested PCR primers 1 and 2R were used to identify inserts. In the 613 colonies we analyzed, 326 had inserts (53.2%), and the insertion size ranged from 200 to 1,200 bp.

The initial RNAi library was constructed with the 326 RNAi plasmids. In the first round of screening, M13 ES cells were transfected with library plasmids individually, and Oct-4 expression levels were measured by quantitative real time RT-PCR 48 hours after transfection. In each group of experiments, the pDC-EGFP plasmid was transfected as a negative control, and pDC-Oct4 and U6shOct4GFP were used as positive controls. The GFP expression cassette in U6shOct4GFP was also used to monitor transfection efficiency. Oct-4 expression level of pDC-EGFP transfected ES cells was taken as the normal value, and the Oct-4 expression levels of library plasmid-transfected ES cells were compared to it. Among first 89 RNAi library plasmids characterized, 12 of them downregulated Oct-4 expression to below 50% of the normal level (Fig. 3A) and 7 plasmids upregulated Oct-4 expression to above 150% of the normal level (Fig. 3B). These 19 plasmids were then transfected into ES cells for a second round of screening. ES cells were selected in medium containing G418 for 7 days, and the pluripotency of resistant colonies was detected by AP staining. In the negative control group transfected by pDC-EGFP plasmid, nearly half of the resistant colonies were morphologically normal and highly positive for AP staining (Fig. 3C, supplemental Table 1), whereas the positive control pDC-Oct4 plasmid significantly reduced the response for AP staining and caused obvious change in colony morphology (Fig. 3D, supplemental Table 1), demonstrating the loss of pluripotency along with differentiation. In the 19 RNAi plasmids picked out from the first round of screening, 4 of them caused cell death. The other 15 plasmids reduced the ratio of AP-positive colonies to less than 60% of the negative control group, notably even to less than 20% by 10 of them. These 10 plasmids were sequenced for the cDNA inserts and in total eight genes were identified (Table 1).) ?) T3 j& j! {8 o! y+ Y5 O

Figure 3. Screening the RNA interference (RNAi) library for self-renewal-related genes. (A, B): Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) results of RNAi library plasmid-transfected ES cells. Real time RT-PCR experiments were performed twice, and SDs were calculated. Oct-4 expression level of pDC-EGFP transfected ES cells was taken as the normal value (defined as 1.0). RNAi library plasmids that downregulated Oct-4 expression to less than 50% of the normal level (A) and upregulated Oct-4 expression to above 150% of the normal level (B) were picked for the second-round screen. (C, D): Alkaline phosphatase staining of ES colonies in the second round of screening after selected for 7 days. Images of negative control group (C) and positive control group (D) were shown as examples. Original magnification is x40.
, x* B% \2 \* O" ?7 y& \
Table 1. Genes identified after two rounds of screening, ]# d) ^$ [3 D2 c6 |1 _
2 J, R) p' P6 y, v4 q- e% h9 `
Functional Analysis of Zfp42/Rex-1 Gene in ES Cell Self-Renewal
8 n8 a3 z; p* s0 ?; H6 V" I* f
To confirm the function of the genes we identified, Zfp42/Rex-1 was chosen for further study. Both knock-down and overexpression assays were carried out to gain further insights into a possible role of Zfp42 in ES cell self-renewal.' d) M3 Z' X& m9 d
8 I" S/ p" z8 _. S% H' \! |
One of three shRNAs designed for Zfp42 RNAi was testified to be highly effective by suppression of the EGFP-Zfp42 fusion protein in Chinese hamster ovary (CHO) cells (supplemental Fig. 2). The effective U6shZfp42 cassette was then cloned into the pEGFP-N1 plasmid to obtain U6shZfp42GFP plasmid. Fortyeight hours after transfection into ES cells, RT-PCR results verified that U6shZfp42GFP plasmid could successfully suppress the expression of the Zfp42 gene (Fig. 4A). Corresponding to the downregulation of Zfp42, expression of Gata6, an endoderm marker, and Fgf5, a marker of a primitive ectoderm, could already be detected (Fig. 4B) before an obvious change of colony morphology (Fig. 5A, 5B), suggesting that the differentiation program had already been triggered. To obtain the long-term effect of Zfp42 RNAi, these transfected ES cells were selected in G418-containing medium for another 5 days, and many resistant colonies formed, but most of them were morphologically differentiated (Fig. 5C, 5D) and negative for AP staining (Fig. 5E). While in the pEGFP-N1 plasmid-transfected control group, most resistant colonies were still morphologically normal and highly positive for AP staining under the same conditions (Fig. 5F). These results clearly show that persistent knock down of Zfp42 could lead to differentiation of ES cells. To further evaluate the nature of the induced differentiation program, the expression of marker genes was examined by RT-PCR (Fig. 4C). A high expression level of parietal and visceral endoderm marker gene Gata4 was detected, indicating differentiation to endoderm lineages. Other marker genes such as Gata6 and Fgf5 as well as the mesoderm marker brachyury (T) were also activated under these conditions. In contrast, expression of trophectoderm marker gene Cdx2 and neuroectoderm marker gene Sox1 were not detected. Hand1, another key transcription factor implicated in trophoblast differentiation was also detected, but only in a considerably late stage (Fig. 4B, 4C), likely because of its expression in mesodermal and neural crest derivatives. Taken together, our data suggested that downregulation of Zfp42 induces ES cells to differentiate to endoderm and mesoderm lineages, consistent with a previous report of differentiation commitment when Oct-4 was upregulated . Our first-round screening results also show that downregulation of Zfp42 would cause upregulation of Oct-4.
1 x4 U. O! M1 M8 G3 F/ m2 I& ?
Figure 4. Expression profiles of Zfp42 knocked down and overexpressed ES cells. (A): Reverse transcription-polymerase chain reaction results of U6shZfp42GFP-transfected ES cells (RNAi) or pEGFP-N1-transfected ES cells as control. ß-Actin was used as internal control, and expression of zfp42 was compared. (B, C): Differentiation marker genes expression profiles of Zfp42 knocked down ES cells at 48 hours after transfection (B) and 7 days after transfection (C). (D): Differentiation marker genes expression profiles of Zfp42 overexpression induced supernatant cells at 7 days after transfection. Abbreviations: Ctr, control; M, marker; RNAi, RNA interference.
) W& m5 A) B+ z; F# d, }1 W
Figure 5. ES cells after knock down or overexpression of Zfp42. (A¨CF): RNA interference knock down of Zfp42 in ES cells. (A, B): Phase contrast and enhanced green fluorescent protein (EGFP) fluorescent image of ES cells at 48 hours after transfection. (C, D): Phase contrast and EGFP fluorescent image of one highly differentiated colony after being selected for 7 days. (E): Alkaline phosphatase (AP) staining of available colonies at 7 days after transfection. (F): AP staining of pEGFP-N1-transfected ES cells under same condition. (G¨CL): Overexpression of Zfp42 in ES cells. Phase contrast and EGFP fluorescent image of ES cells at 24 hours (G, H), 48 hours (I, J), and 72 hours (K, L) after transfection. Original magnification is x100.

To overexpress the Zfp42 gene, plasmid CMV-EGFP-Zfp42, which encodes the fusion protein of EGFP-Zfp42, was constructed. Although several electroporation and Lipofectamine transfection experiments and different conditions were tried, no available colonies could be obtained after 7 days of G418 selection. The states of transfected ES cells were tracked at different stages during the G418 selection. It was very intriguing to find that most GFP-positive cells were floating as small aggregates in the supernatant ever since 24 hours after transfection (Fig. 5G, 5H). At 48 hours after transfection, GFP-positive cells persisted in the supernatant (Fig. 5I, 5J). At 72 hours after transfection, large colonies were also observed, but the cells at the edge of the colonies were morphologically differentiated, and individual cells or groups of cells detached simultaneously from the large colonies (Fig. 5K, 5L). After selecting for 7 days, no available ES colonies other than the floating cells could be obtained. These floating cells could proliferate well in ES medium, G418 selection medium, and EB medium, but their ability to reattach to the culture plate or form EBs was significantly reduced when compared with normally trypsinized ES cells (data not shown). Among the marker genes we examined, Hand1 and Gata4 were expressed in cells harvested from the supernatant after 7 days of G418 selection (Fig. 4D). Therefore, overexpression of Zfp42 also caused ES cells to lose the property of self-renewal. This "detached disaggregation" phenomenon seems to be specific to ES cells, since no similar phenomenon was obtained when the same plasmid was transfected into other mammalian cell lines such as COS-7 and CHO (supplemental Fig. 3). These findings established that maintaining the normal expression level of Zfp42 is important for ES cells self-renewal, and also suggested that Zfp42 may act as a dosage-dependent regulator like Oct-4.
8 X) {$ |' x$ _$ J. N: a6 G! m+ J
DISCUSSION

Methodological Advantages. z7 E' J, N2 `4 n/ ?

A great number of candidate genes are predicted to be involved in ES cell self-renewal, but only very few have been functionally identified. It is impractical to use the traditional homologous recombination-based knock-out method to study gene function on a large scale. As an epigenetic knock-down method, RNAi provides a feasible tool for a large-scale gene function screen. Many successful examples in model organisms have proven the power of this strategy (reviewed in ), but no such work was reported in ES cells until now. Although shRNAi libraries are provided by several companies now, the high financial expense is still a barrier. In contrast, the strategy we established is convenient and cost-saving. The structure of two opposing promoters in pDC plasmid allows a one-step strategy to establish an RNAi library by directly cloning cDNA fragments into pDC backbones. This is also the first report using this kind of RNAi plasmid in ES cells.+ v& ^5 M. v' E5 @9 D
- C2 @1 j. S* H- m! `. T
In large-scale screening work, it is advantageous to select an appropriate subset of candidates first. This step can distinctly improve screen efficiency by eliminating influence of the non-related genes. We are using cDNA subtraction to enrich for potentially related genes. The RNAi library constructed from subtracted cDNAs attained screen efficiency as high as 11% (10/89). Based on PCR technology, cDNA subtraction also provides sufficient resources for large-scale screen. After two rounds of PCR amplification, 326 individual RNAi plasmids were obtained from subtracted cDNAs corresponding to only 1 in 5,000 of total products. An independent experiment revealed that 206 subtracted cDNA fragments correspond to 133 genes and ESTs. Thus only a single subtraction experiment can provide cDNA products that cover 35 times the whole mouse genome., D; i0 K& g+ J" [7 d& q. \

In addition, the two-round screening strategy helps to identify important self-renewal genes. Molecular and cellular techniques are combined to detect both the transient influence and long-term effect of RNAi constructs on ES cells, obtaining a more reliable functional relevance. Among eight candidate genes we identified, two of them (Dppa5, Zfp42) are already predicted to be involved in ES cell self-renewal . And our data further confirmed the important function of Zfp42, it is likely that some of the identified genes will also have direct roles in ES cells self-renewal.

Possible Molecular Mechanism of Zfp42" d/ t& E' B' }
0 d+ o5 E- K' n1 z6 A7 }
The Zfp42 gene is used as a pluripotent marker gene, but no functional relevance has been reported yet . Our data indicated that Zfp42 plays an important role in ES cell self-renewal, and a further bioinformatics assay provides several clues about its possible molecular mechanism.

The Zfp42 gene encodes an acidic zinc finger protein and is predicted to function as a transcription factor . Consistent with this, the predicted binding site of Zfp42 is found at genomic loci near self-renewal genes Oct-4, Nanog, and Zfp42 itself, as well as several differentiation promoting genes such as Cdx2, Gata4, and Hand1. We hypothesize that Zfp42 may function as one of the key regulators in ES cell self-renewal by direct transcriptional regulation of other self-renewal genes and some differentiation-promoting genes (Fig. 6B); along with other interacting factors, a complex regulation network is established. Right now we are using biochemistry methods to determine DNA binding sites and target genes of Zfp42; more evidence will be obtained.
9 ]2 ^; _' T* r
Figure 6. Possible molecular mechanism of Zfp42. (A): Potential dynamic interaction between Zfp42 and Oct-4. Gene expression level is indicated by the intensity of black area. (B): Hypothetical role of Zfp42 in ES cell self-renewal. Abbreviation: RNAi, RNA interference.; R8 r; O/ |4 ~+ p

DISCLOSURES

The authors indicate no potential conflicts of interest.1 R% |/ R" B8 T2 _9 m

ACKNOWLEDGMENTS

We thank Dr. Paul Liu for helping in colony sequencing, Dr. Douglas M. Ruden and Dr. Shawn Burgess for critical reading of our manuscript and editing of English, and Dr. Shuo Lin for helpful discussions. Plasmid U6shOct4GFP is a kind gift from Dr. Duan-Qing Pei, and real time PCR experiments are performed in Dr. Yi Li, Dr. Yu-Xian Zhu, and Dr. Yi-Ping Wang's labs. This work was supported by the Grant "Study of miRNAs' function in mouse ES cells" (number 30470859) and "the Fund for Creative Research Groups" (number 30421004) from the National Natural Science Foundation of China (NSFC). This work was part of the project "Study on a new method of RNA interference" (number 303,40072) supported by NSFC and was also supported by Program for New Century Excellent Talents in University (NCET), the Excellent Young Teachers Program of MO, P. R. C. (EYTP), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (SRF for ROCS, SEM, to B.Z.).
      【参考文献】

* h. H: m4 D' B- K& v; n
Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 2001;17:435¨C462.
( H$ h8 s6 r/ o# w
Smith AG, Heath JK, Donaldson DD et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988;336:688¨C690., b% n: n% W% n! K

Williams RL, Hilton DJ, Pease S et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684¨C687.
5 {, q( `( O% U+ D* B7 R
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281¨C292.
! ^) p$ W8 D2 d, X8 U% j
Sato N, Meijer L, Skaltsounis L, Greengard P et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55¨C63.9 _- ]( [- Z' l& N
  I7 `: I, v( b( g- \  s
Nichols J, Zevnik B, Anastassiadis K et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95:379¨C391.
/ d6 K+ \6 \& n& p% ^
Pesce M, Anastassiadis K, Scholer HR. Oct-4: lessons of totipotency from embryonic stem cells. Cells Tissues Organs 1999;165:144¨C152." w" O7 m# {: w# I' t5 P
! C; m& s$ Y4 V# o9 }" H2 O
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631¨C642.

Chambers I, Colby D, Robertson M et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643¨C655./ K7 z% F. S* z3 j; D

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.
  F4 M# W$ C1 x, n/ B
Rao RR, Stice SL. Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events. Biol Reprod 2004;71:1772¨C1778.

Evsikov AV, Solter D. Comment on " ¡®Stemness¡¯: transcriptional profiling of embryonic and adult stem cells" and "a stem cell molecular signature". Science 2003;302:393b.

Fire A, Xu S, Montgomery MK et al. Potent and specific genetic interference by double-standed RNA in Caenorhabditis elegans. Nature 1998;391:806¨C811.
% S+ W2 Y* ]0 I( w
Yang S, Tutton S, Pierce E et al. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol 2001;21:7807¨C7816.$ w1 h0 ^- d# {# b
5 G7 N" R, Z, B+ F, N
Tang FC, Meng GL, Yang HB et al. Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA. STEM CELLS 2004;22:93¨C99.

Meng GL, Tang FC, Zhang JZ et al. RNA interference in three ES cell lines from different mouse strains. Acta Biochimic et Biophysic Sinica 2003;35:238¨C246.

Grabarek JB, Wianny F, Plusa B et al. RNA interference by production of short hairpin dsRNA in ES cells, their differentiated derivatives, and in somatic cell lines. Biotechniques 2003;l34:734¨C736 739¨C744.

Velkey JM, O'Shea KS. Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells. Genesis 2003;37:18¨C24.: P$ ^0 r3 b- \- \( v& l# p. a! F

Lee Carpenter and Magdalena Zernicka-Goetz. Directing pluripotent cell differentiation using "Diced RNA" in transient transfection. Genesis 2004;40:157¨C163.% z3 N$ b/ n) Y( `' j0 p  M
( B7 V5 p! N; H( P5 I
Hu XL, Shang KG. Establishment and characterization of six ES cell lines from mouse 129/ ter strain. Acta Scientiarum Naturalian Universitatis Pekinensis 1996;32:248¨C253.- u: M; p7 U5 H% @1 s, l

Gunsalus KC, Piano F. RNAi as a tool to study cell biology: building the genome-phenome bridge. Curr Opin Cell Biol 2005;17:3¨C8.3 O- M- E) @" v' e- }, x$ W* C: d2 a

Bierbaum P, MacLean-Hunter S, Ehlert F et al. Cloning of embryonal stem cell-specific genes: Characterization of the transcriptionally controlled gene esg-1. Cell Growth Differ 1994;5:37¨C46.

Tanaka TS, Kunath T, Kimber W.L. et al. Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 2002;12:1921¨C1928.
; [$ f$ g7 |6 H1 b
Hosler Betsy A., Larosa Gregory J., Grippo Joseph F. et al. Expression of REX-I, a Gene Containing Zinc Finger Motifs, Is Rapidly Reduced by Retinoic Acid in F9 Teratocarcinoma Cells. Mol Cell Biol 1989;9:5623¨C5629.

Kaplan T, Friedman N, Margalit H. Ab initio prediction of transcription factor targets using structural knowledge. PLoS Comput Biol 2005;1:e1.3 g0 d) T- T. D
+ Y% }  U" @2 D$ l  E: M& \+ ^
Hosler B A, Rogers M. B, Kozak C. A et al. An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells. Mol Cell Biol 1993;13:2919¨C2928.

Ben-Shushan E, Thompson J. R., Gudas L. J et al. Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site. Mol Cell Biol 1998;18:1866¨C1878.
8 X8 O  C' R9 M1 V
Boyer LA, Lee TI, Cole MF et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005;122:1¨C10./ r/ V. u! h- k$ t

Loh YH, Wu Q, Chew JL et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006;38:431¨C440.

收藏0 分享0 顶0 踩0