Aberrant Genomic Imprinting in Rhesus Monkey Embryonic Stem Cells - 《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 ... 23 下一页

返回列表

查看: 371662\|回复: 225	go Aberrant Genomic Imprinting in Rhesus Monkey Embryonic Stem Cells [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 2009-3-5 00:09 |只看该作者 |倒序浏览 |打印

作者：Akihisa Fujimotoa, Shoukhrat M. Mitalipovb, Hung-Chih Kuoc, Don P. Wolfb作者单位：a Department of Obstetrics  Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan;b Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health  Science University, Beaverton, Oregon, USA;c Stem Cell Program, Institute of Zoology, Genomics Research Center,
   + k! g' D+ c: t6 e# M; [& O$ X
   , f1 P4 E; e6 t7 j6 h

                     # M4 P! ^1 [; Z# G% {
         9 L/ L* E3 @! T* i


            f' O, N1 E; X) ~
                  ; t4 @7 h* m' ]4 O
      2 @4 u$ q, z  ~" q+ _9 ?! K


      【摘要】
   Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction¨Cbased amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.
      【关键词】 Imprinting Embryonic stem cells Rhesus monkey Embryos
   INTRODUCTION9 ?& V9 Y  A  _1 @- K

As many as 200 of the 30,000 genes in the eutherian mammalian genome may be imprinted. In some cases, imprinting is absolute with only one parental allele expressed, whereas for others it is relative depending on developmental time and cell or tissue type .

Embryonic stem cells (ESCs) carry potential for therapies based on cell or tissue replacement. First derived in mice, ESCs are now routinely available in primates, including rhesus macaques and humans . Here, we established the expression patterns of these genes in preimplantation stage embryos generated by ICSI. We then addressed the stability of these imprints in both undifferentiated and in vitro differentiated ESCs. The safety and efficacy of ESC-based treatments should ideally be extensively examined in the nonhuman primate before clinical applications in patients are initiated.+ n# a' B! @3 Q% z0 J7 d

MATERIALS AND METHODS
- h- D0 N7 z2 W0 M1 m- X0 H
Embryo Production and Nucleic Acid Extraction

Controlled ovarian stimulation and oocyte recovery have been described previously . Note that fertilization can be accomplished by conventional in vitro insemination; however, due to our frequent use of cryopreserved sperm, ICSI has become the insemination approach of choice. Briefly, sperm were diluted with 10% polyvinylpyrroli-done (1:4; Irvine Scientific, Santa Ana, CA, http://www.irvinesci.com), and a 5-µl drop was placed in a micromanipulation chamber. A 30-µl drop of TH3 was placed in the same micro-manipulation chamber next to the sperm droplet, and both were covered with paraffin oil (Zander IVF, Vero Beach, FL, http://www.zanderivf.com). The micromanipulation chamber was mounted on an inverted microscope equipped with Hoffman optics and micromanipulators (Olympus, Melville, NY; http://www.olympusamerica.com; Narishige, East Meadow, NY, http://www.narishige.co.jp/niusa). An individual sperm was immobilized, aspirated into an ICSI pipette (Humagen Fertility Diagnostics, Inc., Charlottesville, VA, http://www.humagenivf.com), and injected into the cytoplasm of a metaphase II-arrested oocyte away from the polar body. After ICSI, injected oocytes were placed in four-well dishes (Nalge Nunc International Co., Naperville, IL, http://www.nalgenunc.com) containing protein-free HECM-9 and cultured at 37¡ãC in 5% CO2, 5% O2, and 90% N2. Cultures were maintained under paraffin oil. Embryos at the eight-cell stage were transferred to fresh plates of HECM-9 supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com) and then cultured for a maximum of 9 days with medium changed every other day. Total RNA was extracted from individual blastocysts with Absolutely RNA Nano-prep Kit (Stratagene Co., La Jolla, CA, http://www.stratagene.com) according to the manufacturer¡¯s protocols. When genomic DNA (gDNA) was required from embryos, the first flow-through from the column of Absolutely RNA Nanoprep Kit was used as the starting material for gDNA extraction with a QIAamp DNA Micro Kit (Qiagen, Valencia, CA, http://www1.qiagen.com).: {) n0 x: @: S
- k; ]  @3 o! i& }: [6 H# h. y, b
Monkey ESC Culture and DNA/RNA Extraction
) M  I; p( w' Q8 [2 U9 W
Rhesus monkey ESC culture has been described previously . Briefly, ESCs were grown on feeder layers of mitotically inactivated mouse embryonic fibroblasts (MEFs) in medium consisting of 85% Dulbecco¡¯s modified Eagle¡¯s medium (DMEM)/ F-12 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 15% defined FBS (HyClone), 1 mM L-glutamine (Invitrogen), 0.1 mM µ-mercaptoethanol, and 1% nonessential amino acids (Invitrogen). Medium was changed daily, and ESC colonies were split every 5¨C7 days by manual disaggregation with collected cells replated on fresh MEF layers.1 S+ `9 \" Y6 }( W) Z
9 @) r$ P5 V/ j
For the production of differentiated cells, entire ESC colonies were loosely detached from feeder plates and transferred with ESC medium into ultra-low attachment six-well plates (Corning Inc., Corning, NY, http://www.corning.com). Aggregates were cultured in suspension for 5 days to form embryoid bodies (EBs). To induce further differentiation, EBs were transferred into gelatin-coated culture dishes to allow attachment of cells. After EBs attached and spread out, the medium was replaced with serum-free DMEM/F12 containing 1% ITS supplement (insulin, transferrin, and sodium selenite; Invitrogen) and fibronectin (5 µg/ml; Invitrogen). After 8 further days of culture, medium was replaced with N2 FGF2 medium (DMEM/F12 containing 1% N2 supplement (Invitrogen) and FGF2 (10 ng/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) to induce progenitor cell production. For neuronal phenotypes (serotonin positive, ectodermal lineage), a previously described protocol was employed . Total RNA was extracted from ESCs using RNeasy Mini Kit (Qiagen) according to the manufacturer¡¯s instructions.

PCR-Based Amplification and Allele-Specific Expression Analysis
2 F" b( E9 d6 z" z. r1 P- j- Q
Total RNA from embryos or ESCs was treated with DNAase I before cDNA preparation using SuperScriptTMIII First-Strand Synthesis System for reverse transcription (RT)-PCR (Invitro-gen) according to the manufacturer¡¯s instructions. Characteristics of the SNPs employed for allele-specific expression analysis are summarized in Table 1. For RT-PCR, the following primers were designed to augment those described by us previously for gPCR (Fig. 1 in ).9 D9 P0 z2 i5 X9 J$ E4 |! ?

Table 1. Characteristics of the single nucleotide polymorphisms (SNP) employed for allele-specific expression analysis in rhesus monkey embryos and embryonic stem cells6 `0 z3 V; \4 Y7 }  w) c

Figure 1. Expression analysis of four imprinted and one nonimprinted genes in individual rhesus monkey blastocysts. (A): Restriction fragment length polymorphism (RFLP) analysis with XbaI digestion of NDN amplicons. cDNA from each blastocyst and gDNA from the parents were used as templates. For cDNA, first-round amplification involved Nec-1F/1R followed by second-round amplification with Nec-3F/3R. For gDNA, amplification with only Nec-3F/3R was performed. Amplicons were digested with and without XbaI at 37¡ãC for 16 hours and analyzed by electrophoresis in 1.6% Agarose gels. Bl-1 expressed both parental alleles after Xbal digestion, whereas Bl-2 expressed predominately the maternal allele. (B): Chromatogram showing paternal expression of SNRPN in an informative blastocyst. gDNA from the father was homozygous C/C, whereas maternal gDNA was heterozygous C/T. Blastocyst gDNA was heterozygous with paternal origin of the C allele and maternal origin for the T allele. An expressed C (or paternal) allele was detected in the blastocyst cDNA after reverse transcription¨Cpolymerase chain reaction (RT-PCR) and direct sequencing of the resultant amplicon. The polymorphism site is identified by bold red font. (C): Chromatogram showing IGF2 expression from the paternal allele in an informative blastocyst. gDNA from the father and mother were homozygous T/T and C/C, respectively. An expressed T (or paternal) allele was detected. The polymorphic nucleotide is identified by bold red font. (D): RFLP analysis for an AvaI polymorphism in H19. cDNA of an informative blastocyst and gDNA from both parents were used as templates. For RT-PCR (cDNA), the first round of nested PCR used H19-1F/R primers, whereas the second round of amplification was performed with H19-2F/R primers. For gDNA, only amplification with H19-2F/R was performed. Amplicons were digested with and without AvaI for 16 hours at 37¡ãC and electrophoresed in 3% Agarose gels. This blastocyst (B1) expressed predominately the maternal allele. (E): Chromatogram showing biallelic expression of the nonimprinted gene, GAPD, in an informative blastocyst. The polymorphic nucleotide is identified by bold red font. Abbreviations: Bl-1, blastocyst-1; Bl-2, blastocyst-2; gDNA, genomic DNA; M, molecular ladder., O# g4 e4 r' X. |' \4 G6 O4 Q

Nec-3F; 5'-CGCGAAATCACCAAGCTGCAAATC-3'

Nec-3R; 5'-AGCGAAAGCACCAACCAAGCTTAC-3'
) h( e4 a, p* Z* c+ W4 [5 h
SNRPN-5F; 5'-CCTGGGTGACAAGAGTGAAACTGT-3'3 o( V; t: `7 j( \- e& ?- g

SNRPN-5R; 5'-AGCTCACAAACACTCTACACA-3'
8 X: n! ^) M  U4 j
SNRPN-6R; 5'-TCATACCAGGTGGAGGAGCCATAA-3'
+ m' z' M% \7 w* q. d$ `
H19-2F; 5'-TGAGAGATTCAAAGCCTCCACGAC-3'
9 x( l, `! X4 G! p# Q" T+ O7 b$ F
H19-2R; 5'-CATCAAAGACACCATCGGAACAGC-3'" N+ I4 O& Z2 F9 o. L" n7 S
5 ~+ _0 c; e% _5 H' \  ]
GAPD-2F; 5'-ATCACTGCCACCCAGAAGACT-3'( L- h* ]! _1 m$ F
" N# f& }" ^9 D- w/ T
GAPD-2R; 5'-CACCCTGTTGCTGTAGCCAAATTC-3'  K5 k" Y/ g3 d- E( A& m8 E

Nec-3F/R were the internal primers based on the rhesus monkey sequence of Nec-1F/R. SNRPN primers were based on rhesus monkey exon sequence data from initial screening using gDNA. SNRPN-5F, -5R, and -6R were located in exons 6, 10, and 9, respectively. H19-2F/R were similarly based on the rhesus monkey sequence of H19-F/R. GAPD-2F and GAPD-2R were located on GAPD exon 8 and exon 9, respectively. Their design was based on the human sequence amplifying a 432-bp region in human GAPD transcripts. For NDN, SNRPN, and H19, first-round PCR was carried out in a 20-µl volume containing 2.5 mM MgCl2, 2 mM dNTP mix, 0.4 µM external primer (Nec-1F/R, SNRPN-5F/5R, and H19-1F/R, respectively), and 1.25 units of AccuSure DNA polymerase. PCR conditions were as follows: NDN, 35 cycles at 94¡ãC, 60¡ãC, and 68¡ãC for 30, 60, and 45 seconds, respectively; SNRPN, 35 cycles at 94¡ãC, 60¡ãC, and 68¡ãC for 30, 60, and 45 seconds, respectively; and H19, 35 cycles at 95¡ãC, 62¡ãC, and 68¡ãC for 90, 60, and 45 seconds, respectively. After amplification, 1 µl of the first-round product was transferred to a 19-µl volume containing the same concentration of MgCl2, dNTP mix, DNA polymerase, and internal primers (Nec-3F/R, SNRPN-5F/6R, and H19-2F/R, respectively). PCR conditions were the same as the first amplification. For IGF2, PCR primers and conditions were the same as those used with genomic PCR, except that the number of amplification cycles was increased to 45 to ensure adequate amplification from small amounts of transcript. For GAPD, PCR conditions were 45 cycles at 94¡ãC, 60¡ãC, and 68¡ãC for 30, 60, and 45 seconds, respectively. Expressed alleles were determined by direct sequence analysis of the RT-PCR amplicons or by restriction fragment length polymorphism (RFLP). In all RT-PCR reactions, samples were analyzed without RT in order to exclude the possibility of gDNA contamination. For direct sequence analysis, the same primers used in PCR amplification were employed. RFLP analysis using XbaI and AvaI was available for NDN SNP-1 and H19 SNP-3, respectively . For RFLP, 5 µl of PCR products were digested in a 20-µl volume with 10 IU of restriction enzyme at 37¡ãC for 16 hours, electrophoresed through agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator. Restriction digestion of NDN amplicons with XbaI resulted in 308- and 90-bp fragments from the A-containing allele, whereas the G-containing allele remained uncleaved. Restriction enzyme digestion of H19 amplicons with AvaI resulted in 204- and 46-bp fragments for the C-containing allele, whereas the T-containing allele remained intact.( g( e1 l# w4 |) A; m
: m" [/ n. {* C. [" t$ a7 V
For NDN, IGF2, H19, and GAPD, homozygous parental combinations were used. For SNRPN, only heterozygous parents were available. To examine allele-specific expression of SNRPN in embryos, heterozygosity in gDNA samples was confirmed before RT-PCR analysis. For genomic PCR of SNRPN exon 8 in embryos, a primer located on intron 7 for hemi-nested PCR amplification of exon 8 was designed: SNRPN-5F. For genomic PCR of SNRPN from embryos, first-round amplification using SNRPN-5F/2R was followed by a second amplification with SNRPN-2F/2R. gDNA from ESCs and the parents of the embryos from which the ESC lines were derived were screened for polymorphisms in the transcribed regions of NDN, SNRPN, IGF2, H19, and GAPD as described by us previously . RT-PCR of total RNA extracts of ESC lines that contained informative SNPs, based on parental analysis, generated amplicons that were subjected to sequence analysis.
- u5 g* D4 F2 W; o# z
Quantitative RT-PCR! S. v. R1 l4 t$ ?% E+ Q
# l! l3 v9 R, h- l1 b7 B% }
Real-time RT-PCR was used for the quantitation of allele-specific expression of IGF2 as described by Suda and coworkers  using a custom TaqMan fluorogenic detection system (Custom TaqMan SNP Genotyping Assays; Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). Oregon rhesus monkey embryonic stem cells (ORMES)-1, -6 and -7 were C/T heterozygous for IGF2 SNP-4 and amenable to analysis. RNA extraction and RT was as described above. PCR reaction conditions were 50¡ãC for 2 minutes, 95¡ãC for 10 minutes, 92¡ãC for 15 seconds, and 60¡ãC for 1 minute. The last two steps were conducted for up to 45 cycles. Probe sequences were for the C allele, 5'-VIC-TGACTTCCTG-GTGTG-NFQ-3' and for the T allele, 5'-FAM-TGACTTCTTG-GTGTG-NFQ-3'. C/C and T/T homozygous gDNA samples (5 ng per reaction) were used to generate amplification plots for the 2 IGF2 alleles. Using these allele-specific probes, stable differences in fluorescence (Rn) were seen beyond 40 amplification cycles. A regression coefficient of 0.984 was obtained over a range of gDNA C- to T-allele ratios from 1:8 to 8:1. All PCR reactions were run in triplicate and contained 5 ng of gDNA or cDNA (reverse-transcribed from 10 ng of total RNA). Appropriate negative controls were also run. Real-time PCR was performed on an ABI 7900HT Fast Real-Time PCR System (Applied BioSystems). For each cycle, the threshold cycle (Ct) values for VIC and FAM were determined and the differences in Ct values were used to calculate the ratio of the C to T allele. The averaged Ct based on triplicate determinations was used in all subsequent calculations.9 h7 I8 N, b: t) p' U
' j" ?6 `* W7 V& R& I% p. I
RESULTS

Imprinting in Embryos' E9 F  B; {% j

We first studied blastocysts, because this is the embryonic stage at which implantation occurs and from which ESCs are routinely derived. Animals, heterozygous or homozygous for specific SNPs identified previously . GAPD served as a nonimprinted gene.
: s" F* O) b  n: i" ~! e
Allele-specific expression in NDN was examined using a G/A polymorphism (SNP-1) for which three animal pairs were homozygous, G for the male and A for the female, and 27 expanded blastocysts were examined individually. Of the 24 expressing embryos, 22 were biallelic and two showed maternal expression (Fig. 1A). Blastocyst ages varied between 7 and 9 days, but there was no apparent correlation between NDN expression and age. With SNRPN, nine embryos were analyzed from an animal pair in which the male was C homozygous and the female C/T heterozygous (SNP-11 and primers SNPRN 5F/6R). Genomic PCR revealed that three embryos were C/T heterozygous, and subsequent sequence analysis from their cD-NAs indicated expression from the C, or paternal, allele (Fig. 1B). IGF2 expression was examined in five informative blastocysts from two animal pairs carrying T/C polymorphisms (SNPs-1 and 4). Only the paternal allele was expressed (Fig. 1C). For H19, six heterozygous blastocysts were examined from two animal pairs that were homozygous for a T/C polymorphism (SNP-3). Expression from the maternal allele was observed (Fig. 1D). Finally, using an A/G polymorphism in one parental combination (SNP-1), we recovered two embryos and confirmed biallelic expression of GAPD (Fig. 1E).

Imprinting in Undifferentiated ESCs' a0 i, O" |) t, p# A

Next, we defined imprinting in the undifferentiated ESC phenotype. Several ESC lines derived from ICSI-produced embryos were available in the ORMES series  in ORMES-1, -6, and -8 but observed no differences.

Figure 2. Allele-specific expression analysis in undifferentiated and differentiated rhesus monkey ESC (ORMES-6) populations. The polymorphic nucleotide is identified in chromatograms by bold red font. (A): Chromatograms showing paternal expression of NDN in ORMES-6. The paternal gDNA was homozygous, C/C, whereas the maternal and ESC gDNAs were heterozygous C/T. The C allele in the ESC line must have a paternal origin, whereas the T allele was derived from the mother. cDNA from undifferentiated and neuronally differentiated ESC populations contained only the C (or paternal) allele. (B): Chromatograms showing paternal expression of SNRPN in ORMES-6 based on a G/A polymorphism. (C): Chromatograms showing biallelic expression of IGF2. (D): Chromatograms showing biallelic expression of GAPD. (E): Restriction fragment length polymorphism (RFLP) analysis of H19 showing biallelic expression in undifferentiated and in one neuronally differentiated (OR6N1, passage 24) cell population, and maternal in another (OR6N3, passage 28). Abbreviations: ESC, embryonic stem cell; gDNA, genomic DNA; ORMES, Oregon rhesus monkey embryonic stem cells.
- c4 U' u: h6 M* y4 M* f
Table 2. Summary of allele-specific expression studies in undifferentiated populations from ORMES cell lines0 x3 m' |; J$ u- i; Y6 B

We also applied quantitative RT-PCR to confirm the ratios of biallelic expression of IGF2 in ORMES-1, -6, and -7. Averaged values (¡ÀSD) of the paternal- to maternal-allele ratio were 0.86 ¡À 0.1, 0.98 ¡À 0.05, and 0.84 ¡À 0.28, respectively (Fig. 3).4 `* M2 [( _% L1 \+ V1 N1 n3 b

Figure 3. Quantitative polymerase chain reaction analysis of IGF2 allele-specific expression in undifferentiated and differentiated cell populations for ORMES-1, -6, and -7 cell lines. Each sample was run at least in triplicate. Undifferentiated = ; neuronal =  pancreatic = .5 Z5 T, T7 g2 ~( r

Imprinting in Differentiated ESCs
; ]9 {0 M% h4 H0 A8 n9 R
Finally, anticipating that therapies involving ESCs will use progenitor or terminally differentiated progeny, allele-specific expression in neural or pancreatic phenotypes was examined. ESCs were differentiated into neural progenitor populations that were more than 70% nestin and Musashi 1¨Cpositive , up to 70% of the treated cells were serotonin-positive. Similarly, between 67% and 78% of exendin-4¨C and nicotinamide-treated cells were C-peptide¨Cpositive (results not shown). OCT4 expression was negative, confirming the absence of undifferentiated cells. NDN transcripts were examined in ORMES-6¨Cderived neuronal populations at passages 24 and 26. Complimentary DNA showed the presence of the C (or paternal) allele, indicating normal paternal expression (Fig. 2A; Table 3). For SNRPN, ORMES-6¨Cderived differentiated cells at passages 24 and 26 expressed the paternal allele (Fig. 2B; Table 3). These results show that monoallelic expression of NDN and SNRPN was maintained after differentiation. IGF2, which was not imprinted in undifferentiated ORMES lines, was also expressed from both parental alleles in differentiated cell populations (Fig. 2C; Table 3). Expression of the silent maternal allele in IGF2 was confirmed by real-time PCR analysis for both neuronal and pancreatic phenotypes in ORMES-1, -6, and -7 (Fig. 3). Paternal- to maternal-allele ratios varied from 1.11 to 1.47 for neuronal populations and from 1.16 to 1.35 for pancreatic populations. Significant differences (9 e. W: D! A, ^8 D* g2 `
" ^) r3 _3 B# A  h
Table 3. Summary of expression analysis of four imprinted genes and one nonimprinted gene in rhesus monkey blastocysts, ESCs, and ESC-derived neuronal cell populations
. D5 j) I3 E, l, c
Monoallelic expression (maternal) of H19 in ORMES-5 was preserved in neuronal cell populations (results not shown). In contrast, the biallelic expression of H19 seen in undifferentiated ORMES-6 was retained in neuronal cell populations examined from passage 24, whereas monoallelic (maternal) expression occurred in cells from passage 28 (Fig. 2E). GAPD was expressed from both parental alleles in ORMES-6¨Cderived neuronal cells (Fig. 2D)." O  g/ L- a  f' p4 O1 U; n
! F3 \! u) C3 Z
DISCUSSION

The assisted reproductive technologies have facilitated the development of invasive procedures from ICSI to preimplantation genetic diagnosis, somatic cell nuclear transfer, and ESC derivation. Studies in mice suggest that preimplantation embryos taken from the natural environment and exposed to the stress induced by in vitro culture or manipulation can lead to disruption of epigenetic marks at imprinted loci, resulting in aberrant growth and morphology at fetal and perinatal stages of development .2 b6 L5 k7 K/ F
, t2 E3 z; t+ W- g) P
In monkey blastocysts, NDN was expressed either from the maternal allele or from both alleles. This result could arise from expression of residual maternal mRNA, an unlikely possibility given the expectation that most maternal transcripts are absent by the time of embryonic gene activation .: e9 T1 w! ^4 g, W

In monkey ESC lines, NDN and SNRPN showed paternal expression similar to the adult; however, we observed loss of monoallelic expression of H19 and IGF2 with paternal- to maternal-allele ratios for IGF2 in the range of 0.84 to 1.64. Interestingly, human embryonic germ .2 g1 r2 h% p- C7 g6 |

Differentiated ESCs retained the expression pattern seen in undifferentiated cells with the exception of H19, in which expression varied between maternal and biallelic as a function of passage number. This variability could represent the sampling of different cell populations in a given culture or in the cell phenotypes present at different passage numbers.
+ O9 x  L* F- B. @% ^
The significance of imprint loss in monkey ESCs to cell function and fate after transplantation of progeny into a host animal remains unknown. Epigenetic instability in murine ESCs is not incompatible with normal development, because murine ESCs can support the routine generation of normal chimeric mice and even apparently healthy cloned animals with gene expression abnormalities that are not severe enough to impede development to birth , epigenetic regulation of cell growth is a subject that must be considered in greater detail before clinical stem cell use is attempted.
9 k$ z* B9 I' q, r
DISCLOSURES
6 y0 I. d  }$ S" P; C& I# u
The authors indicate no potential conflicts of interest.
0 i6 Q; y% B- k# V" O
ACKNOWLEDGMENTS

We thank Lisa Clepper for assistance with molecular biology techniques, Cathy Ramsey, Carrie Thomas, Michelle Sparmann, and the ART core for management of animals, Yibing Jia for sequencing, and Drs. James Byrne and Jon Hennebold for critical review of the manuscript. This work was supported by the National Institutes of Health.
      【参考文献】
0 l0 S! h* u7 {' a# E

Ohlsson R, Hedborg F, Holmgren L et al. Overlapping patterns of IGF2 and H19 expression during human development: Biallelic IGF2 expression correlates with a lack of H19 expression. Development 1994;120: 361¨C368.! ^  c5 C1 d4 r4 k6 y. A& [

Vu TH, Hoffman AR. Promoter-specific imprinting of the human insulin-like growth factor-II gene. Nature 1994;371:714¨C717.

Bartolomei MS, Tilghman SM. Genomic imprinting in mammals. Annu Rev Genet 1997;31:493¨C525.- J; ^! O% {, B" g! T* z4 K% Y
0 S. R" }$ E& O) U6 X5 u: N  T
Dean W, Santos F, Stojkovic M et al. Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A 2001;98:13734¨C13738.: V5 n) E8 L" F7 S
( N& A  _; y4 Y( b/ F& W" s0 @
DiBerardino MA. Genomic Potential of Differentiated Cells. New York: Columbia University Press, 1997./ b6 q: y. d$ W0 y8 @

Feinberg AP, Cui H, Ohlsson R. DNA methylation and genomic imprinting: Insights from cancer into epigenetic mechanisms. Semin Cancer Biol 2002;12:389¨C398.

Thompson SL, Konfortova G, Gregory RI et al. Environmental effects on genomic imprinting in mammals. Toxicol Lett 2001;120:143¨C150.& v" Z# e2 N1 L8 D; A
5 q( g# M7 t# h
Lucifero D, Chaillet JR, Trasler JM. Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology. Hum Reprod Update 2004;10:3¨C18.

Nicholls RD, Knepper JL. Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes. Annu Rev Genomics Hum Genet 2001;2:153¨C175.
5 R/ b+ y/ X4 i/ {* u) t3 _9 k
Mann MR, Bartolomei MS. Towards a molecular understanding of Prader-Willi and Angelman syndromes. Hum Mol Genet 1999;8:1867¨C1873.1 R' H/ p1 H- l. o8 V1 g
' H# ~3 K0 c5 G* I& X& x0 `1 s" F
Weksberg R, Smith AC, Squire J et al. Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum Mol Genet 2003;12:R61¨C68.+ o! j* Q1 d% _9 a# [3 [
8 ?, V( w- M0 X4 `
Doherty AS, Mann MR, Tremblay KD et al. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod 2000;62:1526¨C1535.( e5 q" f; c1 j" v( z. L

Maher ER, Brueton LA, Bowdin SC et al. Beckwith-Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet 2003; 40:62¨C64.

Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844¨C7848.

Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145¨C1147.
- \& q' d8 q6 B! q  W6 B- k
Fujimoto A, Mitalipov SM, Clepper L et al. Development of a monkey model for the study of primate genomic imprinting. Mol Hum Reprod 2005;11:413¨C422.2 B% |9 ^! ]: Q9 Z1 ]% I
& @8 N( D8 r: \$ P' O
Wolf DP, Thormahlen S, Ramsey C et al. Use of assisted reproductive technologies in the propagation of rhesus macaque offspring. Biol Re-prod 2004;71:486¨C493.
, w8 d8 p. q# U
McKiernan SH, Bavister BD. Culture of one-cell hamster embryos with water soluble vitamins: Pantothenate stimulates blastocyst production. Hum Reprod 2000;15:157¨C164.* W! e7 u2 x4 v
  g! {! ^; S# j& p- ^+ s9 f% m
Schramm RD, Paprocki AM, VandeVoort CA. Causes of developmental failure of in-vitro matured rhesus monkey oocytes: Impairments in embryonic genome activation. Hum Reprod 2003;18:826¨C833.0 a" M0 A* M- O9 D5 c
; j, V2 k0 |" o7 _: @
Mitalipov SM, Nusser KD, Wolf DP. Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos. Biol Reprod 2001; 65:253¨C259.
, [+ L1 v+ H- o6 e% v5 s& ~
Kuo HC, Pau KY, Yeoman RR et al. Differentiation of monkey embryonic stem cells into neural lineages. Biol Reprod 2003;68:1727¨C1735.

Salli U, Reddy AP, Salli N et al. Serotonin neurons derived from rhesus monkey embryonic stem cells: Similarities to CNS serotonin neurons. Exp Neurol 2004;188:351¨C364.4 S" A  g' e' C8 y. H  w

Lester LB, Kuo HC, Andrews L et al. Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes. Reprod Biol Endocrinol 2004;2:42.
1 m- z& `8 F. W" L- S
Mitalipov SM, Kuo HC, Hennebold JD et al. Oct-4 expression in pluri-potent cells of the rhesus monkey. Biol Reprod 2003;69:1785¨C1792.

Suda T, Katoh M, Hiratsuka M et al. Use of real-time RT-PCR for the detection of allelic expression of an imprinted gene. Int J Mol Med 2003;12:243¨C246.

Schumacher A, Doerfler W. Influence of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem cells. Nucleic Acids Res 2004;32:1566¨C1576.
! x' n- f, O/ P1 F4 a3 U$ B8 X" r
Dean W, Santos F, Reik W. Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin Cell Dev Biol 2003;14:93¨C100.

Mitalipov SM, Kuo HC, Johnson JA et al. Isolation of rhesus monkey embryonic stem (ES) cell lines, efficiency, karyotype stability and growth characteristics. San Francisco: International Society for Stem Cell Research, 2005:1661¨C1162. Poster 1547.7 b- g1 b. H* r( Q1 y2 V+ y( e! H5 Z
! B* x9 c( @1 O3 [, \1 o
Rideout WM 3rd, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science 2001;293:1093¨C1098./ ~5 z5 X+ i4 b
, T6 n* J& P, N, D+ t2 {" _7 \' h
Humpherys D, Eggan K, Akutsu H et al. Epigenetic instability in ES cells and cloned mice. Science 2001;293:95¨C97.
0 B3 `7 |7 y7 L3 V# G( Y- l
Dean W, Bowden L, Aitchison A et al. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: Association with aberrant phenotypes. Development 1998;125:2273¨C2282.

Gosden R, Trasler J, Lucifero D et al. Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet 2003;361: 1975¨C1977.

Lighten AD, Hardy K, Winston RM et al. IGF2 is parentally imprinted in human preimplantation embryos. Nat Genet 1997;15:122¨C123.

Jinno Y, Sengoku K, Nakao M et al. Mouse/human sequence divergence in a region with a paternal-specific methylation imprint at the human H19 locus. Hum Mol Genet 1996;5:1155¨C1161.0 _1 b; J/ W- c1 Q& D2 Z% r- T' `; w
1 `$ B9 J3 t, t
Salpekar A, Huntriss J, Bolton V et al. The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos. Mol Hum Reprod 2001;7:839¨C844.

Schultz RM. The molecular foundations of the maternal to zygotic transition in the preimplantation embryo. Hum Reprod Update 2002;8: 323¨C331.- G! V  e3 B1 p2 V  V
$ E2 ^& T/ `% o  ]1 y1 `
Hanel ML, Lau JC, Paradis I et al. Chromatin modification of the human imprinted NDN (necdin) gene detected by in vivo footprinting. J Cell Biochem 2005;94:1046¨C1057.

Huntriss J, Daniels R, Bolton V et al. Imprinted expression of SNRPN in human preimplantation embryos. Am J Hum Genet 1998;63:1009¨C1014.6 B( t8 v. b$ W, h7 C: \
1 ^! x) |# ?, u, \" W/ }  z
Ren J, Lee S, Pagliardini S et al. Absence of Ndn, encoding the Prader-Willi syndrome-deleted gene necdin, results in congenital deficiency of central respiratory drive in neonatal mice. J Neurosci 2003;23:1569¨C1573.
* q) B! Q6 s9 R5 G2 v" ?8 R0 _8 H
Onyango P, Jiang S, Uejima H et al. Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages. Proc Natl Acad Sci U S A 2002;99:10599¨C10604.

Rugg-Gunn PJ, Ferguson-Smith AC, Pedersen RA. Epigenetic status of human embryonic stem cells. Nat Genet 2005;37:585¨C587.; f* _# K( }: e" d" j
. z, g! x) N# v, l) {( j
Cui H, Niemitz EL, Ravenel JD et al. Loss of imprinting of insulin-like growth factor-II in Wilms¡¯ tumor commonly involves altered methylation but not mutations of CTCF or its binding site. Cancer Res 2001; 61:4947¨C4950.

Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature 1993;366:362¨C365.& v% v# {. e6 j7 h5 ]! n. G- p8 {
3 ^3 W1 h$ Y+ _) D
Wang Z, Meissner A, Jaenisch R. Nuclear cloning and epigenetic reprogramming. In: Lanza R, Gearhart J, Hogan B et al., eds. Handbook of Stem Cells: Embryonic, Vol. 1. London: Elsevier Academic Press, 2004: 119¨C127.

收藏0 分享0 顶0 踩0