Development of a monkey model for the study of primate genomic imprinting.
ABSTRACT An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT-PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.
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ABSTRACT: We propose an algorithm creating consistent, dense disparity maps from incomplete disparity data generated by a conventional stereo system used in a wide-baseline configuration. The reference application is IBR-oriented immersive videoconferencing, in which disparities are used by a view synthesis module to create instantaneous views of remote speakers consistent with the local speaker's viewpoint. We perform spline-based disparity interpolation within nonoverlapping regions are defined by discontinuity boundaries identified in the incomplete disparity map. We demonstrate very good results on significantly incomplete disparity data computed by a conventional correlation-based stereo algorithm on a real wide-baseline stereo pair acquired by an immersive videoconferencing system.Pattern Recognition, 2004. ICPR 2004. Proceedings of the 17th International Conference on; 01/2004
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ABSTRACT: Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.Stem Cell Research 07/2011; 7(1):28-40. · 4.47 Impact Factor
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ABSTRACT: Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources—inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia—as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.Russian Journal of Developmental Biology 11/2008; 39(6):325-336. · 0.22 Impact Factor
Development of a monkey model for the study of primate
A.Fujimoto1, S.M.Mitalipov2, L.L.Clepper2and D.P.Wolf2,3
1Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan and2Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University,
Beaverton, OR 97006, USA
3To whom correspondence should be addressed at: Department of Reproductive Sciences, Oregon National Primate Research Center,
505 NW 185th Avenue, Beaverton, OR 97006, USA. E-mail: email@example.com
An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers
that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2,
known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres
(ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene.
Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was
isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons
resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since
disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipu-
lation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or
by ICSI. Muscle biopsies followed by RT–PCR and sequence analysis were performed in four heterozygous animals produced
by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally
imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific
studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated
embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differen-
tially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells
of the monkey.
Key words: development/imprinted genes/methylation/primate/single nucleotide polymorphisms
The majority of genes in the mammalian genome are equally
expressed from both maternal and paternal alleles. A small number
(,50) are imprinted; epigenetically regulated and inherited in a
silent state from one of the two parents and in a fully active form
from the other (Rideout et al., 2001; Surani, 2001). With some, the
epigenetic mark is not absolute but rather is either partial, tissue-
specific, cell-type specific or restricted to certain developmental
stages. Most imprinted genes show allelic differences in DNA meth-
ylation at so-called differentially methylated regions (DMRs) which
act as epigenetic modifiers of allelic expression by recruiting pro-
teins that specifically bind to methylated or unmethylated regions
(Bell and Felsenfeld, 2000; Hark et al., 2000; Schoenherr et al.,
2003). Imprinting centres (IC) are regulatory sequences that harbour
We have been interested in imprinted gene expression in the
embryos and embryonic stem (ES) cells of rhesus monkeys for sev-
eral reasons. First, the monkey represents a clinically relevant
species to study the early developmental events that are precluded
for ethical reasons in the human. Access to preimplantation stage
embryos and ES cell lines in the rhesus macaque is now routine
(Wolf, 2004; Wolf et al., 2004a) and the major ethical limitations
associated with human studies can be largely circumvented because
of the close phylogenetic relationship between monkey and man.
Second, given the potential of ES cell-based therapies to be used in
human medicine, an understanding of the role(s) of imprinted genes
in the function of ES cell progeny both in vitro and in vivo is essen-
tial. Questions concerning the integrity of imprinting in undifferen-
tiated and differentiated progeny can only be addressed fully in an
animal model. Third, some imprints are altered in response to
environmental insults (e.g., H19 and IGF2 in the mouse) (Sasaki
et al., 1995; Dean et al., 1998; Doherty et al., 2000; Khosla et al.,
2001; Mann et al., 2003) or to in vitro manipulations that include
nuclear transfer (Zhang et al., 2004) and ICSI. The use of ICSI for
the treatment of human subfertility has been associated with
increased rates of Beckwith–Wiedemann syndrome, likely reflecting
aberrant imprinting (Maher et al., 2003). Furthermore, abnormal
expression of the imprinted genes, NDN and SNRPN, has been
implicated in the aetiology of Prader-Willi syndrome (Reis et al.,
1994; Mann and Bartolomei, 1999) while IGF2 and H19 abnormal-
ities have been associated with Beckwith–Wiedemann syndrome
and Wilms’ tumours (Moulton et al., 1994; Weksberg et al., 2003).
In order to evaluate the role of imprinted genes in primate devel-
opment, suitable genetic markers are required. Presently, despite
Molecular Human Reproduction Vol.11, No.6 pp.413–422, 2005
Advance Access publication May 20, 2005doi:10.1093/molehr/gah180
q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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widespread use of rhesus macaques in biomedical research,
relatively little is known about the organization or content of the
genome in this species, a predicament that will change in the near
future with the development of a genetic linkage map (J. Rogers;
personal communication), whole genome DNA sequencing (www.
hgsc.bcm.tme.edu/projects/macaque/) and the commercial avail-
ability of an Affymetrix microarray for expression analysis (rhesus-
genechip.unomaha.edu/index.html). Regardless, we have begun an
investigation of SNPs in the monkey genome that are relevant to
determining the parent-specific expression and methylation status of
imprinted genes; those localized to the transcribed regions of several
candidate genes, including NDN, H19, SNRPN and IGF2 or within
postulated ICs and positioned either upstream of H19 or in the
promoter-exon 1 region of SNURF/SNRPN. Here, we report the defi-
nition of 32 SNPs useful to allele-specific expression and methyl-
ation analysis in the rhesus macaque. Evidence was obtained that
these four genes are imprinted in the monkey and that the expected
uniparental expression of NDN, H19, SNRPN and IGF2 was retained
in infants produced from in vitro manipulated embryos. Further-
more, our studies demonstrated that CpG islands within monkey
H19/IGF2 and SNURF/SNRPN ICs were differentially methylated
suggesting that CpG methylation could be one of the mechanisms
involved in the differential marking of parental alleles in monkey
gametes. This approach lays the foundation for detailed studies on
the role of imprinting in a clinical relevant primate model.
Materials and methods
Adult animals selected from the colony maintained at Oregon National
Primate Research Centre (ONPRC) and assigned for reproductive studies
were accessed. Indian-derived animals (10 males and 45 females) were
descendants of animals imported in the 1970s and raised at ONPRC.
Chinese-origin females (38) were imported to ONPRC in the last 3–4 years
as captive-reared from south China, Vietnam, Cambodia and Laos (G. Heck-
man, personal communication). The number of animals tested varied across
loci reflecting the frequency of the particular SNP under study. All animal
procedures were approved by the Institutional Animal Care and Use Com-
mittee at the ONPRC.
Genomic DNA extraction and amplification by PCR
Genomic DNA was isolated from peripheral blood leukocytes using a
QIAamp DNA Blood Midi Kit (QIAGEN, Valencia, CA) according to the
manufacturer’s protocol. The following primers for genomic PCR were
based on human consensus sequences obtained from GenBank. The relative
positions of the human primers and polymorphic sites are shown in Figure 1.
In one instance, H19-1R, the original human-based primer sequence was
replaced by the monkey sequence when it became available in order to
improve amplification efficiency. Two of these imprinted genes, IGF2 and
H19, are located on chromosome 11 in both the human and rhesus monkey.
NDN and SNRPN are located on chromosome 15 in the human which corre-
sponds to chromosome 7 in the rhesus monkey (Lawce et al., 1998).
Figure 1. Schematic diagram showing relative positions of primers and reported human polymorphisms for NDN, H19, IGF2, SNRPN and GAPD used in the
identification of SNPs in the rhesus monkey. Numbered rectangles represent human exons and dotted areas denote coding regions. The asterisks indicate fre-
quent polymorphic nucleotides reported in the human. The barred area in IGF2 exon 9 represents a CA repeat region.
A.Fujimoto et al.
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PCR reactions were carried out in a 20ml volume containing 2.5mM
MgCl2, 2mM dNTP mix, 0.4mM each primer, 40ng template DNA and 1U
of AccuSure DNA polymerase (Bioline, Randolph, MA). PCR reactions for
CTCF6-SNP-F/R were carried out using 0.2mM each primer, 250ng of tem-
plate gDNA and 45ml of Platinum PCR Supermix High Fidelity (Invitrogen,
Carlsbad, CA) containing a final concentration of 2.16mM MgSO4,
0.198mM dNTPs. PCR conditions were as follows (denaturation/annealing/
extension): for NDN-1F/R, 35cycles 94/60/688C for 30/60/45s; for NDN-
2F/R, 35 cycles 94/60/688C for 30/60/45s; for SNRPN-1F/R, 35 cycles
94/65/688C for 30/60/45s; for SNRPN-2F/R, 35 cycles 94/65/688C for
30/60/45s; for SNRPN-3F/R, 35 cycles 94/65/688C for 30/60/45s; for
SNRPN-4F/R, 35 cycles 94/65/688C for 30/60/45s; for IGF2-1F/R, 35 cycles
94/65/688C for 30/60/45s; for H19-1F/R, 35 cycles 95/62/688C for
90/60/45s; for GAPD-1F/1R, 35cycles 94/65/688C for 30/60/45s, for
CTCF6-SNP-F/R, 35 cycles of 94/58.9/688C for 30/60/60s; for SNRPN-
DMR–F/R, 35 cycles of 94/61/688C for 30/60/45s. Amplicons were electro-
phoresed through 1.6% TBE agarose gels stained with ethidium bromide and
visualized on a UV transilluminator.
PCR products were purified (QIAquick Gel Extraction Kit, QIAGEN) and
sequenced with an ABI 3100 capillary genetic analyzer (Applied Biosystems,
Foster City, CA) using BigDye terminator sequencing chemistry (Wen,
2001). Sequencing results were analysed using software Sequencher (Gene
Codes Corporation, Ann Arbor, MI).
Restriction enzyme digestion
To create restriction fragment length polymorphisms (RFLP), 5ml of PCR
products were digested in a 20ml volume with 10IU of restriction enzyme at
378C for 16h and digested samples were electrophoresed through agarose
gels, stained with ethidium bromide and visualized on a UV transilluminator.
Restriction enzymes were selected based on specificity using a Webcutter 2.0
RFLP (online software from Yale University http://www.firstmarket.com/
cutter/cut2.html). XbaI and AvaI were appropriate for NDN SNP-1 and H19
Frequency was determined by the formula; 2 £ the number of animals homo-
zygous for the allele þ the number of heterozygous animals divided by 2£
the number of animals tested (Zhang et al., 2004).
Total RNA extraction from muscle biopsy tissues and RT–PCR
Monkeys were anesthetized with Propofol and approximately 20mg of
muscle tissue was removed from the quadriceps femoris using a 16-gauge
biopsy needle (Jorgensen Laboratory Inc., Loveland, CO).
Total RNA was isolated by using TRIzolw (Invitrogen) according to the
manufacturer’s protocol. To eliminate DNA contamination, samples were
treated with preamplification-grade DNase I (Invitrogen). RT was performed
with a Superscript III system (Invitrogen) and PCR reactions were done for
each sample with and without (negative control) RT. PCR protocols were the
same as those described above for genomic DNA in NDN, H19, SNRPN and
IGF2. Regarding the GAPD (glyceraldehyde-3-phosphate dehydrogenase)
gene, exon primers GAPD-2F/2R were used to amplify a polymorphic region
in the amplicon with intron primers, GAPD-1F/1R. PCR conditions were 35
cycles of 94/60/688C for 30/60/45s.
Amplicons were sequenced as described above for genomic DNA samples.
Expressed alleles were determined from sequence analysis of the amplicons
generated from genomic DNA of both parents and their infants and compli-
mentary DNA from skeletal muscle tissue.
Bisulphite modification of DNA and methylation-specific PCR
Genomic DNA was extracted from muscle biopsy samples using a DNeasy
Tissue Kit (QIAGEN) and approximately 2mg of gDNA was modified by
busulphite treatment using a CpG Genome Modification Kit (Chemicon
International, Temecula, CA) according to the manufacturer’s protocols.
Primers for methylation-specific PCR (MSP) were designed using online
software (http://www.urogene.org/methprimer/) as described elsewhere (Li
and Dahiya, 2002). MSP primers were:
Bi-H19-DMR methyl F; 50-GTATAAGAATTCGGAGATTTTTGC-30
Bi-H19-DMR methyl R; 50-GTAAACCCTACGACACCTAACGTA-30
Bi-H19-DMR unmethyl F; 50-TATAAGAATTTGGAGATTTTTGTGT-30
Bi-H19-DMR unmethyl R; 50-CATAAACCCTACAACACCTAACATA-30
SNRPN bi methyl F; 50-GGT ATT GGG ATT TTT GTA TTG CGG-30
SNRPN bi methyl R; 50-ACG CAA CTA ACC TTA CTC GC-30
SNRPN bi unmethyl F; 50-GGT ATT GGG ATT TTT GTA TTG TG-30
SNRPN bi unmethyl R; 50-ACA CAA CTA ACC TTA CTC ACT C-30
PCR reactions were carried out in a 50ml volume containing 1.5mM
MgCl2, 0.2mM each dNTP, 0.25mM each primer, 5ml modified DNA and
2.5U of Platinum Taq DNA Polymerase, (Invitrogen). PCR conditions were;
for Bi-H19-DMR methyl F/R and Bi-H19-DMR unmethyl F/R, 35 cycles of
94/51/728C for30/30/45s; for SNRPN
of 94/53/728C for 30/30/45s; for SNRPN bi unmethyl F/R, 35 cycles of
94/49/728C for 30/30/45s. Amplicons were electrophoresed through 1.6%
TBE agarose gels stained with ethidium bromide and visualized on a UV
bimethylF/R, 35 cycles
Detection of single nucleotide polymorphisms in NDN,
SNRPN, IGF2, H19 and GAPD
In order to define SNPs in rhesus monkeys that could be exploited
in allele-specific expression studies, we first selected appropriate pri-
mers based on consensus human DNA sequences. These primers
were used to amplify, by PCR, the sequence of interest in genomic
DNA isolated from peripheral blood leukocytes. PCR products were
then purified and sequenced for individual animals. In this manner,
we discovered 2 SNPs in NDN, 3 in H19, 13 in SNRPN, 4 in IGF2,
and 1 in GAPD, a non-imprinted gene. The GenBank accession
numbers for these monkey sequences along with sites and base
substitutions for all SNPs are described in Table I. The percent
homology of these monkey amplicons to the human sequence varied
from 90.4 to 97.1%.
In the case of NDN, overlapping PCR amplicons covering 914bp
that were 564 and 361bp in length, respectively, resulted from the
use of two primer pairs, 1F/1R and 2F/2R. This region contained
two novel SNP sites, a G to A polymorphism at nucleotide position
135 and a T to C transition at position 795 in exon 1 (Table I).
Using primer pair H19-1F/1R, a PCR amplicon consisting of
525bp was sequenced with the identification of three SNPs; a G to
A polymorphism at position 358; a G to A transition at 439 and a T
to C transition at 457 in exon 5.
The primer pair, SNRPN-1F/1R, amplified a 338bp region con-
taining one SNP, a G to A transition at position 132 while SNRPN-
2F/2R amplified a 273bp region containing T to C polymorphisms
at positions 83 and 95 (including exon 8 in the human). Overlapping
PCR amplicons covering 1042bp resulted from the use of SNRPN-
3F/3R and SNRPN-4F/4R which were 564 and 505bp in length,
respectively. Nine SNPs were identified in this sequence (Table I).
With IGF2, no evidence of a monkey SNP in the polymorphic
region in exon 9 identified in Figure 1 in the human was obtained.
However, the primer pair IGF2-1F/1R amplified a 471bp sequence
containing four polymorphic sites in exon 9, downstream of a CA
repeat region. These sites included T to C polymorphisms at 169,
286 and 405bp and an A to C conversion at position 371.
SNPs for gene expression analysis in macaques
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As a non-imprinted gene control, we used GAPD. GAPD-1F and
GAPD-1R amplified a 517bp product putatively covering the region
from intron 7 to the exon 8/intron 8 boundary. A single SNP was
identified as a G to A polymorphism at position 358.
Distribution of SNPs in Indian-derived versus Chinese-
Genomic DNA from up to 93 animals was screened depending on
the SNP under study and the results have been summarized as a
function of animal origin. Substrain differences in tested animals
were seen in several SNPs (Tables II and III). For SNP NDN-1, the
majority of animals tested were homozygous, either G/G (45) or
A/A (7), the former presumably reflecting the wild-type. In 23 of
the 75 animals, a G/A heterologous condition existed. This poly-
morphism showed a significant difference in allele frequency by
Chi-square analysis dependent on the animal’s country of origin; it
was only found in Indian-origin animals. Similarly, for SNPs in
H19, only Indian-origin animals showed the polymorphisms.
Although SNPs 1-13 in the SNRPN tended to be expressed most
often in Chinese-origin animals, allele frequency for the non-domi-
nant allele was low. There were no examples of SNPs in IGF2 that
were significantly different as a function of the substrain, although
the number of tested animals was low. The SNP identified in GADP
was defined in 21 animals and found to be present in both Indian-
and Chinese-origin monkeys with an overall genotype distribution
of 42.9/28.6/28.6 for A/A, G/G and A/G, respectively. An allele fre-
quency of 57% A versus 43% G was statistically indistinguishable
from the 50:50 ratio expected for a non-imprinted gene. The results
in Tables II and III provide valuable insights required for the selec-
tion of animals to be used in allele-specific expression studies.
The imprint status of NDN, H19, IGF2 and SNRPN in
The genes selected for study, NDN, H19, IGF2 and SNRPN are
known to be imprinted in mice, cattle and humans (Bartolomei et al.,
1991; DeChiara et al., 1991; Leff et al., 1992; Giannoukakis et al.,
1993; Zhang et al., 1993; Reed and Leff 1994; MacDonald and
Wevrick, 1997; Zhang et al., 2004). Preliminary results from gene
expression profiling of primate parthenogenetic stem cells suggest
that at least NDN and SNRPN as paternally expressed genes are also
imprinted in the cynomolgus macaque (Hipp et al., 2004). We
examined the status of these genes in rhesus monkey somatic
(skeletal muscle) tissues with the expectation that if they were
imprinted, we would see differential expression of the two alleles in
In four infants produced by natural mating, heterozygosity in
their genomic DNA samples for the SNPs listed in Table I were
found; two for NDN, one for H19, two for SNRPN, three for IGF2,
and two for GAPD.
Using the primers NDN-1F/R, PCR and direct sequence analysis
of the resulting amplicon, we confirmed G/A heterozygosity in
female 24063. The cDNA isolated from muscle in this animal and
amplified using the same primer set showed expression of only the
G allele, the outcome expected for an imprinted gene (Figure 2A).
Similarly, female, 24073, was heterozygous (C/T) for H19-SNP-3
and cDNA analysis from the muscle tissue biopsy showed
expression of only the T allele (Figure 2D).
Table I. Primers, site and base substitution for SNPs in the rhesus monkey
SNPPCR primers Geneback accession numberBP positiona
Base substitution type
aRelative to the sequence of the monkey amplicon.
A.Fujimoto et al.
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Female, 23985, was heterozygous (A/G) for both SNRPN-SNP-10
and IGF2-SNP-2 (C/T). The cDNA from her muscle cells expressed
only the A and T alleles, respectively, again indicating that these
two genes are imprinted in the monkey (Figure 2B and C).
Finally, female, 24063, who was also heterozygous (G/A) for
GAPD-SNP-1, showed evidence of biallelic expression of this non-
imprinted gene in the cDNA from her muscle (Figure 2E).
Expressed alleles examined in other informative animals showed
the expected monoallelic expression of all four imprinted genes and
biallelic expression of GAPD (Table IV). These results strongly
suggest that these four genes are imprinted in muscle cells of mon-
keys, although it was not possible to determine which parental allele
was expressed without characterizing both parents.
Analysis of allele-specific expression in ICSI
We began to address the potential impact of oocyte/embryo manipu-
lation on imprinting in the monkey by examining allele-specific
expression in somatic cells. This undertaking is especially relevant
to H19 that has been shown to be unstable in mice (Sasaki et al.,
1995; Dean et al., 1998; Doherty et al., 2000; Khosla et al., 2001;
Mann et al., 2003) and cattle (Zhang et al., 2004). Genomic DNA
was isolated from the blood cells of gamete donors for ICSI pro-
duced infants (Wolf et al., 2004b) and was screened for the SNPs in
Tables I–III. Based on parental analysis, five ICSI-infants were
selected and genomic DNA from their blood samples was screened
for the presence of heterozygosity in possibly informative genes. As
screening primers, we used NDN-1F/R, SNRPN-4F/R, IGF2-1F/R,
H19-1F/R and GAPD-1F/R. With NDN and SNRPN, we selected
primers based on the availability of RFLP (NDN-SNP-1) and the
frequency of the SNP (SNRPN-SNP-5-13).
In five ICSI produced monkeys, allele-specific expression analysis
was possible; two for NDN, four for SNRPN, two for IGF2, four for
H19 and four for GAPD.
Analysis of genomic DNA from female, 24089, and her parents
indicated that she was informative for allele-specific expression of
all four imprinted genes. Direct sequence analysis of the amplicon
produced with NDN-1F/1R identified this female as heterozygous
for the G to A polymorphism located at 135bp (NDN-SNP-1),
while the paternal and the maternal genomic DNA samples were
homozygous for G and A, respectively, at the same polymorphic
Table II. Allele and genotype frequencies of NDN, H19, SNRPN and IGF2 SNPs in Indian-origin rhesus monkeys
n (%) of each
SNPs for gene expression analysis in macaques
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