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Allelic switching of the imprinted IGF2R gene in cloned bovine fetuses and calves
Animal Reproduction Science 116 (2009) 19–27
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Allelic switching of the imprinted IGF2R gene in cloned bovine fetuses and calves T. Suteevun-Phermthai a,c,1, C.L. Curchoe a,∗,1, A.C. Evans b, E. Boland b, D. Rizos b, T. Fair b, P. Duffy b, L.Y. Sung a, F. Du a, S. Chaubal a, J. Xu a, T. Wechayant c, X. Yang a, P. Lonergan b, R. Parnpai c, X.C. Tian a a
Department of Animal Science and Center for Regenerative Biology, University of Connecticut, Storrs, CT 06269, USA Department of Animal Science, Faculty of Agriculture, University College Dublin, Belfield, Ireland c Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand b
a r t i c l e
i n f o
Article history: Received 28 August 2008 Received in revised form 5 January 2009 Accepted 14 January 2009 Available online 20 January 2009 Keywords: Large Offspring Syndrome cloned cattle IGF2 receptor gene Imprinted genes
a b s t r a c t Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3’UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells
∗ Corresponding author. E-mail address: [email protected] (C.L. Curchoe). 1 The authors contributed equally to this work. 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.01.003
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to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Imprinted genes are expressed in a parent-of-origin specific manner and many function as fetal growth regulators (Rotwein and Hall, 1990; Moore and Haig, 1991). In the developing fetus, genetic imprints are established during gametogenesis and are maintained in somatic cells but undergo erasure and parent-of-origin specific re-establishment in primordial germ cells. The exact nature of genetic imprints is still unclear; however, most imprinted genes contain regions of parental specific differential methylation. The imprinting phenomenon is believed to be unique to placental mammals, putatively due to the placenta’s status as the maternal–fetal interface and driven by the so-called maternal/paternal “tug-of-war” (Moore and Haig, 1991). Indeed, numerous imprinted genes are only expressed or imprinted in the placenta and many differences in the regulation of imprinted genes have been found among mammals with different placental types. For instance, the insulin-like growth factor 2 receptor (IGF2R) has been found to be imprinted in mice (Willison, 1991), sheep (Young et al., 2001), cattle (Killian et al., 2001) and swine (Killian et al., 2001) but is polymorphically imprinted in humans (Wutz and Barlow, 1998). To date, more than 80 imprinted genes have been identified in the mouse, many of which have orthologues in other species. The imprinted IGF2R is highly conserved between mice and cattle (Killian et al., 2001). The IGF2R gene is maternally expressed and functions to suppress fetal growth by acting as a scavenger receptor for the imprinted fetal mitogen IGF2. In mice, the mono-allelic expression of IGF2R is believed to be regulated through an antisense transcript, antisense IGF2R (AIR) whose promoter is located in intron 2 of IGF2R (Sleutels et al., 2002). Large Offspring Syndrome (LOS) and failure to develop to term are observed in many animal species cloned to date, most notably in cloned mice and cattle (Wakayama and Yanagimachi, 1999; Hill et al., 2000; Tamashiro et al., 2000; Tanaka et al., 2001). The relatively lesser efficiency of live healthy births from somatic cell nuclear transfer (SCNT) could be due to the differences in epigenetic establishment of imprinted genes between SCNT and conventional reproduction. In somatic cell nuclear transfer, differentiated somatic cells, with pre-existing genetic imprints, are used to generate cloned animals. Contrasting that, gametes participating in conventional reproduction have already undergone imprint erasure and re-establishment. To date, molecular characterization of cloned mice and cattle has revealed that the allelic expression of imprinted genes of the somatic donor cells is transmitted to clones. In a previous study, cloned calves and age-matched controls were generated at day 25 (before implantation), day 45 (during implantation) and day 75 (after implantation) (Lonergan et al., 2007). Signs of LOS were observed starting from day 45 of gestation. In the present study, (1) allelic expression pattern of IGF2R in the placentas of day 25 and day 45 fetuses, (2) tissue-specific IGF2R allelic expression and relative amount of expression in day 75 cloned and control fetuses, (3) IGF2R antisense transcript expression, AIR, in all cloned and control fetuses, and (4) whether IGF2R gene expression varies from controls in spontaneously aborted cloned fetuses and calves that died shortly after birth compared with clones that were born healthy, were assessed. 2. Materials and methods 2.1. Tissue samples, extraction of genomic DNA and total RNA Cloned fetuses at gestation day 25, day 45 and day 75 were generated by SCNT using skin fibroblasts from an elite Holstein–Friesian dairy cow. This donor cow was chosen because she was heterozygous
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for a previously described expressed SNP in IGF2R (Yang et al., 2005). This SNP was used to monitor IGF2R allelic expression. Organ samples were also obtained from three cloned beef cattle fetuses aborted near term (approximately 252 day in gestation), term placentas from four full-term cloned beef calves that died shortly after birth (approximately 280 day) and two living beef calf clones. These beef calf clones were generated from fibroblasts of a 13 year-old beef steer (Parnpai, 2004). Control organ/placenta samples for the day 25, day 45 and day 75 clones were generated using in vivo produced embryos by superovulation (Lonergan et al., 2007). Control tissues for aborted fetuses, term clones and surviving clones were obtained from tissues of cattle at a slaughterhouse. Organ/tissue samples included the extra-embryonic tissues, cotyledon, inter-cotyledonary placental membrane (intercotyledon), brain, heart, kidney, liver, lung, and spleen. All organ samples were frozen immediately after collection and stored at −80 ◦ C until analysis. Genomic DNA and total RNA were extracted from organ samples using the DNeasy and RNeasy kits from Qiagen (Valencia, CA), respectively. The extracted RNA was treated with RNase-free DNase to remove any possible contaminating genomic DNA. The Institutional Animal Care and Use Committee at the University of Connecticut and an equivalent organization at the Institute of Agricultural Technology (Thailand) approved all procedures involving the use of animals in the US and Thailand. All experimental procedures involving animals in Ireland were approved by the Animal Research Ethics Committee at University College Dublin and were licensed by the Department of Health and Children, Ireland, in accordance with the cruelty to animals act (Ireland 1897) and European Community Directive 86/609/EC. 2.2. Genotyping for heterozygosity of an SNP in the 3’ UTR of cattle IGF2R To genotype all animals used in the present study, genomic DNA was amplified using PCR primers designed to flank an SNP within the 3 -untranslated region (UTR) of the cattle IGF2R gene; 5 -AGCCAAACAAGAGTACAAA-3 (forward) and 5 -AGAAGCCTTAATTTGCACA-3 (reverse). DNA sequencing, according to standard sequencing methods, confirmed the G/A polymorphism. PCR was performed using 100 ng of genomic DNA with 0.6 pmol/l of each primer and Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA) in a total volume of 25 l. The following amplification conditions were used: an initial denaturation step of 94 ◦ C for 3 min was followed by 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 58 ◦ C for 20 s, and extension at 72 ◦ C for 50 s, and a final extension at 72 ◦ C for 7 min. PCR products were analyzed on a 1% agarose gel before screening for heterozygosity by single strand conformational polymorphism (SSCP) as described previously (Curchoe et al., 2005). Briefly, denatured PCR products were resolved on a 12% polyacrylamide gel at 4 ◦ C for 20 h. The polyacrylamide gel was then silver-stained (Bosari et al., 1995), dried and archived. The presence of two bands confirmed the heterozygosity of the samples. 2.3. Allele-specific expression of the imprinted cattle IGF2R gene For all samples identified as heterozygous for the IGF2R SNP, one step RT-PCR was performed using the one-step RT-PCR enzyme mix (Qiagen). RNA was reverse transcribed using the following conditions: 50 ◦ C for 30 min and 95 ◦ C for 15 min before PCR amplification using the conditions described above. The RT-PCR products were then analyzed for allelic expression by SSCP as described above. 2.4. Quantitative real-time RT-PCR of cattle IGF2R Total RNAs (20 ng) from tissues collected at day 25, day 45 and day 75 of gestation were reversetranscribed at 50 ◦ C for 1 h with Superscript III (Invitrogen). The real time RT-PCR reaction mixture contained 5 l of cDNA, 12.5 l of SYBR green master mix (Applied Biosystems Inc., CA, USA) and 0.3 m of forward and reverse primers in a total volume of 25 l. All tissues were analyzed in triplicate for every gene. The specificity of the real time RT-PCR product was proven by a melting curve analysis. To compare the relative amounts of gene expression in different samples, the comparative cycle (CT) method, also known as the 2−CT method, was applied as we described previously, using the following formula: CT = CTsample − CTcalibrator , where CT = threshold cycle for the target gene
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amplification, CT = CTtarget gene − CTendogenous reference . The endogenous reference gene chosen for this study was the ˇ-actin gene. The calibrator was a mixture of RNA from liver and lung tissues and was included in each real time amplification reaction. All validation and quantification were conducted as suggested by the manufacturer of the real time amplification kit (Applied Biosystems Inc.). Validation of the amplification efficiency of target genes and the endogenous reference gene was completed before using the 2−CT method for quantification. Briefly, different dilutions of cDNA (range tested) were amplified by real time RT-PCR using the gene specific primers (IGF2R, 5 -CCGGGAGATGGTAATGAGCA-3 (forward) and 5 -TCTCGTTCTCGTCGGCCT-3 (reverse) and -actin, 5 -ACCGTGAGAAGATGACCCAGA-3 (forward) and 5 -TCACCGGAGTCCATCACGAT-3 (reverse)). The corresponding CT values were plotted against the log of each cDNA amount and the data were plotted using linear regression analysis. The values of the slopes of the validation curves for each gene was less than 0.1, indicating that the same amplification efficiency was obtained for all samples with both greater and lesser amounts of the cDNA. 2.5. Expression of the bovine IGF2R antisense transcript, AIR RT-PCR primers were designed to amplify an intronic region between IGF2R exons 1 and 2, thereby ensuring that only the IGF2R antisense transcript, AIR would be amplified. The sequences of the forward and reverse primers were 5 -AGAGCGTGGCTGAGTCTGACG-3 and 5 -CGAGACCCCACCAGACTAGAC-3 . Total RNA was amplified with the Qiagen One-Step RT-PCR kit with 1000 ng of total RNA, 0.6 pmol/l primers and 2 l reverse transcriptase in a total volume of 25 l at 55 ◦ C for 30 min. 2.6. Statistical analysis Differences in the relative amounts of mRNA of IGF2R in different organs between cloned and control animals were analyzed using the Kruskal–Wallis subroutine (a non-parametric alternative to one-way independent sample ANOVA) of the Statistical Analysis System (SAS) and a probability value of p ≤ 0.05 was considered to be significant. 3. Results 3.1. Genotyping for bovine IGF2R and presumptive parental allele determination All day 25, day 45, day 75, aborted, dead and live clones and age-matched control cattle were confirmed for heterozygosity of an SNP in the 3’UTR of the IGF2R gene. A specific band of 298 bp was amplified and sequenced. A G/A transition was identified at position 52 (Fig. 1(A)). The heterozygous and homozygous patterns are revealed by SSCP as two and one band for each sample (Fig. 1(B)). The G allele was preferentially expressed in the fibroblast and cumulus cells of the donor Holstein–Friesian dairy cow used to create the day 25, day 45 and day 75 clones (Yang et al., 2005) (data not shown). The A allele was preferentially expressed in the donor cells of the beef animal used to create the aborted, living and cloned animals that died shortly after birth (Fig. 1(B)). Due to the lack of parental tissues for the donor animals, the parental origin of the G and A alleles could not be determined. However, based on the fact that the maternal allele is the expressed allele in naturally reproduced cattle, it was assumed that the G and A alleles are the maternal alleles in Holstein and beef cattle donor animals, respectively. 3.2. Allele-specific expression of the imprinted IGF2R gene in clones and controls In control animals, one allelic expression pattern of IGF2R was observed in all tissues, including extra-embryonic tissues at day 25 (Table 1) and day 45 (Table 1) of gestation, organ tissues collected at day 75 of gestation (Table 2) and from newborn calves (Table 3). This gene expression pattern was consistent with predominate expression of the presumptive maternal allele as indicated by the darker band with a lesser amount of “leaky” expression of the presumptive imprinted/paternal allele, as indicated by the lighter band (Fig. 1(B)). The only exception to this finding was observed in the brains
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Fig. 1. (A) Sequence chromatogram of PCR product shows the SNP, a G to A transition, at the bp 52 position (arrow) of the cattle IGF2R gene. (B) SSCP genotyping of the SNP in the 3’UTR of the cattle IGF2R gene. Lanes 1–3, represent a heterozygous (G/A) sample and homozygous (AA) and (GG) samples, respectively. Lanes 4–9, demonstrate different patterns derived from all samples; Lane 4, the heterozygous (AG) pattern; Lane 5, allelic expression of the donor cells used to create the clones in Table 1, showing mostly allele A expression with leakage of the G allele; the A allele is assumed to be the maternal allele. Lanes 6 and 8, display examples of homozygous GG allelic expression, Lanes 7 and 9 display examples of homozygous AA allelic expression.
of newborn controls, were the IGF2R gene was bi-allelically expressed, i.e. the two bands representing the expression of the maternal and paternal alleles were equal in intensity (Fig. 1(B)). Because this is the first detailed characterization of allelic expression in conventionally bred cattle, the “leaky” imprinting pattern is assumed to be the normal pattern of IGF2R gene expression in cattle in all tissues with the exception of the newborn brain, which is bi-allelic.
Table 1 Allelic expression patterns of the IGF2R gene in the trophectoderm of day 25 and day 45 controls and clones. Capital letters represent the parental allele with the strongest expression (M = maternal, P = Paternal). Extra-embryonic tissues
Day 25
Day 45
Controls Clone #1 Clone #2 Clone #3 Clone #4
pM ” ” ” ”
pM ” ” ” P
Symbol (”) indicates no difference in allelic expression from the control. Table 2 Allelic expression patterns of the IGF2R gene in organs/tissues of day 75 control and cloned fetuses. Capital letters represent the parental allele with the strongest expression (M = maternal, P = Paternal). Day 75
Controls Clone #1 Clone #2 Clone #3 Clone #4
Heart
Brain
Liver
Spleen
Cotyledon
Intercotyledon
pM ” ” ” ”
pM ” ” ” ”
pM ” ” ” ”
pM ” P PM PM
pM ” ” ” ”
pM ” ” ” ”
Symbol (”) indicates no difference in allelic expression from controls.
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Table 3 Allelic expression patterns of the IGF2R gene in living clones, clones which died shortly after birth (approximately 280 day), aborted clones, near-term fetuses (approximately 250 day) and fetuses derived from conventional breeding. Capital letters represent the parental allele with the strongest expression (M = maternal, P = Paternal). Approximate Day 250–280
Controls Living #1 Living #2 Living #3-6 Aborted #1 Aborted #2 Aborted #3 Dead #1 Dead #2 Dead #3 Dead #4
Brain
Cotyledon
Heart
Kidney
Liver
Lung
Placenta
MP
Mp MP MP ” ” P P ” ” ” MP
Mp
Mp
Mp
Mp
P P MP ” ” MP MP
mP P MP ” ” MP mP
P P M ” ” ” mP
MP P MP ” ” mP mP
Mp ” ” ” P M P ” ” ” ”
P M ” ” ” ”
Symbol (”) indicates no difference in allelic expression from controls. Blank areas indicate that tissues were not collected.
In cloned fetuses, the normal “leaky” imprinting pattern of IGF2R in cattle was observed in all extraembryonic tissues collected at day 25, and 3 out of 4 extra-embryonic tissues collected at day 45 of gestation. One of the day 45 extra-embryonic tissue displayed complete monoallelic gene expression without “leakage” (Table 1). However, the expressed allele was the putative paternal allele which normally only displays “leaky” gene expression. While most organs of day 75 cloned fetuses had normal expression patterns, Clone 1 was also switched to exclusive expression of the paternal allele in the spleen. In Clones 3 and 4, the spleen also displayed the abnormal biallelic expression pattern of IGF2R (Table 2). The most variable allelic expression patterns, out of all the age groups studied, were observed in spontaneously aborted cloned calves. Every tissue tested could display exclusive switching (no “leakage”) to the paternal allele (Table 3) in different cloned animals. Only one instance of normal allelic gene expression was observed in this group (in the cotyledon of aborted clone 1). Several abnormal instances of exclusive maternal expression, skewing to the paternal allele and equal expression of both alleles were also observed in this group. Two cases of skewing toward the paternal allele were observed in the group of clones that died shortly after birth, one in the kidney, liver and lung of one calf and one in the lung of another (Table 3). The brains and placentas of clones that died shortly after birth and the placentas of living clones all displayed the same allelic expression as those of the controls. Representative SSCP gels are shown in Fig. 2. 3.3. Quantitation of IGF2R expression Amounts of IGF2R gene expression were measured in all tissues of day 75 controls and cloned animals (Fig. 3) and in the trophectoderm of day 25 and day 45 cloned and control animals. In fetuses produced by conventional reproduction, the spleen is the major producer of IGF2R, while the liver, lung, kidney, and placenta produce intermediate amounts of IGF2R. In samples from the present study, the brain and heart produced negligible amounts of IGF2R. This pattern of tissue-specificity was maintained in cloned animals. Interestingly, the expression of the IGF2R gene in all of the tissues was similar for cloned fetuses and control fetuses with the exception of the spleen, for which the cloned fetuses had lesser amounts when compared to control fetuses. Despite the lack of significant differences between controls and clones in the steady state amounts of IGF2R gene expression in most tissues, individual clones displayed more or less in a number of tissues, as evidenced by the large standard error bars for the clones (Fig. 3). For instance, day 75, clone 3 displayed 26 and 11% of IGF2R gene expression (relative to controls) in the spleen and lung, respectively, while showing 462% more than controls in the liver. In one instance (day 75, clone 2) IGF2R gene expression was consistently less, displaying 55,
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Fig. 2. Representative SSCP gels of the allelic expression of IGF2R in organs/tissues of living clones, clones which died shortly after birth, aborted clones and at day 25,day 45 and day 75 of gestation. (A) Lanes 1–4: representative gel of alleles AG, AA, GG and the donor cell pattern, respectively, Lanes 5–8: representative of the leaky pattern seen in the heart, liver, lung and spleen of newborn control fetuses. (B) Lanes1–3: representative image of normal “leaky” gene expression observed in the heart, kidney and liver of a cloned calf that died shortly after birth, Lanes 4 and 5: representative image of the normal biallelic expression seen in the brain of cloned cattle that died shortly after birth. Lanes 6–8: representative image of the switched mono-allelic expression seen in the heart, kidney and liver of an aborted cloned calf. The data for all cloned animals are summarized in Table 1.
27, 8 and 1% expression of IGF2R in the spleen, cotyledon, lung and brain, respectively, compared to the average amounts in the corresponding tissues in controls. The trophectoderm of day 25 and day 45 cloned fetuses showed no significant difference in amounts of gene expression when compared to controls of the same age group. The placentas of the living clones showed normal “leaky” expression of IGF2R.
Fig. 3. Relative amounts of IGF2R gene expression as folds over the calibrator for organs/tissues of day 75 cloned (solid bar) and control (open bar) animals. The symbol (*) represents a significant difference of p < 0.05.
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Fig. 4. Representative agarose gel image AIR expression in all tissues tested in the organs/tissues of control and cloned cattle at day 75 of gestation. Lane 1: 1 kb + ladder, Lane 2: negative control, Lanes 3–8: brain, cotyledon, kidney, liver, lung, inter cotyledon of a control animal, Lanes 8–11: inter cotyledon, lung and liver of a cloned animal.
3.4. Expression of the IGF2R antisense transcript, AIR All control and cloned organ/tissue samples expressed AIR as detected by RT-PCR (Fig. 4). No difference was observed in AIR expression in tissues expressing different IGF2R alleles. 4. Discussion In the present study, the IGF2R allelic expression pattern and amounts of gene expression throughout development of the placenta, were established by examining day 25 trophectoderm (before implantation), 45 trophectoderm (during implantation) and 75 placenta and cotyledons (after implantation), and in live healthy term cattle by SSCP and real time RT-PCR. Allelic expression was found to be monoallelic with “leakage” of the imprinted allele in day 25, day 45, day 75 and term cattle placental structures. Age-matched placental structures from clones were compared to controls and no differences were found in the relative amounts of IGF2R gene expression between day 25 and day 45 cloned and control trophectoderm using the Kruskal–Wallis subroutine of the SAS. When comparing the rate of change between the day 25 and day 45 trophectoderm to that of the control group, no differences were found. While no overall differences were found in the cotyledon and intercotyledon of day 75 fetuses between clones and controls, individual clones had a large amount of variation, such as (day 75) clone 2, which had 60% of IGF2R gene expression in the cotyledon compared to the control group. These data are consistent with the results of Lonergan et al. (2007) whose data have suggested that large calf syndrome manifests after day 45 of gestation. The allelic expression pattern of IGF2R in the cotyledon and intercotyledon area of healthy, live cloned cattle was consistent with the data from the control cotyledon and inter cotyledon area, with most allelic expression patterns showing monoallelic expression with “leaky” expression of the imprinted allele. In sharp contrast to this the placentas and cotyledons of spontaneously aborted cattle displayed abnormal monoallelic expression of the maternal allele or allele switching to the paternal allele. Normal “leaky” expression of the IGF2R gene was not observed in this group of samples. IGF2R allelic expression patterns and amounts of gene expression in the organs of day 75 cloned and control fetuses were highly variable in some individual clones compared to age matched controls. No differences in amounts of IGF2R gene expression were found in most organs, when taken together as a group, and the allelic expression data correlates well with this observation. Most day 75 organ samples display monoallelic expression with “leakage”, which compares well with the data from Yang et al. (2005). However, the expression of the IGF2R gene in the spleen was found to be different when compared to the control group, which correlates well with the allelic expression data observed in the present study. Two instances of monoallelic, paternally switched expression were observed and most other samples appeared biallelic, instead of displaying the typical “leaky” expression pattern. Organs from cloned calves that died shortly after birth display normal “leaky” gene expression patterns, similar to age matched controls. Organs from spontaneously aborted fetuses, however, had the greatest variability in IGF2R gene expression patterns. Every organ tested was capable of display-
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ing abnormal allelic expression in different animals. Only one case of normal allelic expression was observed in this entire group (the cotyledon of aborted clone #1). The observed allelic switching was not due to a lack of AIR expression because AIR was detected in all tissues examined, including those in which allele switching occurred. Normally, AIR is expressed from the paternal allele, and it silences the expression of the paternal IGF2R by interfering with the initiation of IGF2R’s paternal promoter. It is unclear if, in the tissues that expressed the paternal allele of IGF2R, AIR is also switched to the maternal allele because we did not have a polymorphism to investigate this possibility. Further studies can explore the regulation of gene expression in tissues displaying allele switching. Because less expression of the IGF2R gene in one organ does not predict less expression in other organs (with the notable exception of day 75 clone #2), it is possible that an epigenetic abnormality may have occurred early in development before the onset of organogenesis, as suggested by Smith et al. (2005). Taking these data together, proper allelic expression of IGF2R has a role in terms of sustaining a typical period of gestation, as evidenced by the group of aborted fetuses, which displayed the greatest allelic irregularities, in contrast to the group of day 75 cloned fetuses, which displayed the least allelic irregularities. It is concluded that proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Acknowledgements The authors would like to thank Hamed Kian and Yuqin Zhang for control tissue collection from the slaughterhouse. 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Allelic switching of the imprinted IGF2R gene in cloned bovine fetuses and calves T. Suteevun-Phermthai a,c,1, C.L. Curchoe a,∗,1, A.C. Evans b, E. Boland b, D. Rizos b, T. Fair b, P. Duffy b, L.Y. Sung a, F. Du a, S. Chaubal a, J. Xu a, T. Wechayant c, X. Yang a, P. Lonergan b, R. Parnpai c, X.C. Tian a a
Department of Animal Science and Center for Regenerative Biology, University of Connecticut, Storrs, CT 06269, USA Department of Animal Science, Faculty of Agriculture, University College Dublin, Belfield, Ireland c Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand b
a r t i c l e
i n f o
Article history: Received 28 August 2008 Received in revised form 5 January 2009 Accepted 14 January 2009 Available online 20 January 2009 Keywords: Large Offspring Syndrome cloned cattle IGF2 receptor gene Imprinted genes
a b s t r a c t Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3’UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells
∗ Corresponding author. E-mail address: [email protected] (C.L. Curchoe). 1 The authors contributed equally to this work. 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.01.003
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to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Imprinted genes are expressed in a parent-of-origin specific manner and many function as fetal growth regulators (Rotwein and Hall, 1990; Moore and Haig, 1991). In the developing fetus, genetic imprints are established during gametogenesis and are maintained in somatic cells but undergo erasure and parent-of-origin specific re-establishment in primordial germ cells. The exact nature of genetic imprints is still unclear; however, most imprinted genes contain regions of parental specific differential methylation. The imprinting phenomenon is believed to be unique to placental mammals, putatively due to the placenta’s status as the maternal–fetal interface and driven by the so-called maternal/paternal “tug-of-war” (Moore and Haig, 1991). Indeed, numerous imprinted genes are only expressed or imprinted in the placenta and many differences in the regulation of imprinted genes have been found among mammals with different placental types. For instance, the insulin-like growth factor 2 receptor (IGF2R) has been found to be imprinted in mice (Willison, 1991), sheep (Young et al., 2001), cattle (Killian et al., 2001) and swine (Killian et al., 2001) but is polymorphically imprinted in humans (Wutz and Barlow, 1998). To date, more than 80 imprinted genes have been identified in the mouse, many of which have orthologues in other species. The imprinted IGF2R is highly conserved between mice and cattle (Killian et al., 2001). The IGF2R gene is maternally expressed and functions to suppress fetal growth by acting as a scavenger receptor for the imprinted fetal mitogen IGF2. In mice, the mono-allelic expression of IGF2R is believed to be regulated through an antisense transcript, antisense IGF2R (AIR) whose promoter is located in intron 2 of IGF2R (Sleutels et al., 2002). Large Offspring Syndrome (LOS) and failure to develop to term are observed in many animal species cloned to date, most notably in cloned mice and cattle (Wakayama and Yanagimachi, 1999; Hill et al., 2000; Tamashiro et al., 2000; Tanaka et al., 2001). The relatively lesser efficiency of live healthy births from somatic cell nuclear transfer (SCNT) could be due to the differences in epigenetic establishment of imprinted genes between SCNT and conventional reproduction. In somatic cell nuclear transfer, differentiated somatic cells, with pre-existing genetic imprints, are used to generate cloned animals. Contrasting that, gametes participating in conventional reproduction have already undergone imprint erasure and re-establishment. To date, molecular characterization of cloned mice and cattle has revealed that the allelic expression of imprinted genes of the somatic donor cells is transmitted to clones. In a previous study, cloned calves and age-matched controls were generated at day 25 (before implantation), day 45 (during implantation) and day 75 (after implantation) (Lonergan et al., 2007). Signs of LOS were observed starting from day 45 of gestation. In the present study, (1) allelic expression pattern of IGF2R in the placentas of day 25 and day 45 fetuses, (2) tissue-specific IGF2R allelic expression and relative amount of expression in day 75 cloned and control fetuses, (3) IGF2R antisense transcript expression, AIR, in all cloned and control fetuses, and (4) whether IGF2R gene expression varies from controls in spontaneously aborted cloned fetuses and calves that died shortly after birth compared with clones that were born healthy, were assessed. 2. Materials and methods 2.1. Tissue samples, extraction of genomic DNA and total RNA Cloned fetuses at gestation day 25, day 45 and day 75 were generated by SCNT using skin fibroblasts from an elite Holstein–Friesian dairy cow. This donor cow was chosen because she was heterozygous
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for a previously described expressed SNP in IGF2R (Yang et al., 2005). This SNP was used to monitor IGF2R allelic expression. Organ samples were also obtained from three cloned beef cattle fetuses aborted near term (approximately 252 day in gestation), term placentas from four full-term cloned beef calves that died shortly after birth (approximately 280 day) and two living beef calf clones. These beef calf clones were generated from fibroblasts of a 13 year-old beef steer (Parnpai, 2004). Control organ/placenta samples for the day 25, day 45 and day 75 clones were generated using in vivo produced embryos by superovulation (Lonergan et al., 2007). Control tissues for aborted fetuses, term clones and surviving clones were obtained from tissues of cattle at a slaughterhouse. Organ/tissue samples included the extra-embryonic tissues, cotyledon, inter-cotyledonary placental membrane (intercotyledon), brain, heart, kidney, liver, lung, and spleen. All organ samples were frozen immediately after collection and stored at −80 ◦ C until analysis. Genomic DNA and total RNA were extracted from organ samples using the DNeasy and RNeasy kits from Qiagen (Valencia, CA), respectively. The extracted RNA was treated with RNase-free DNase to remove any possible contaminating genomic DNA. The Institutional Animal Care and Use Committee at the University of Connecticut and an equivalent organization at the Institute of Agricultural Technology (Thailand) approved all procedures involving the use of animals in the US and Thailand. All experimental procedures involving animals in Ireland were approved by the Animal Research Ethics Committee at University College Dublin and were licensed by the Department of Health and Children, Ireland, in accordance with the cruelty to animals act (Ireland 1897) and European Community Directive 86/609/EC. 2.2. Genotyping for heterozygosity of an SNP in the 3’ UTR of cattle IGF2R To genotype all animals used in the present study, genomic DNA was amplified using PCR primers designed to flank an SNP within the 3 -untranslated region (UTR) of the cattle IGF2R gene; 5 -AGCCAAACAAGAGTACAAA-3 (forward) and 5 -AGAAGCCTTAATTTGCACA-3 (reverse). DNA sequencing, according to standard sequencing methods, confirmed the G/A polymorphism. PCR was performed using 100 ng of genomic DNA with 0.6 pmol/l of each primer and Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA) in a total volume of 25 l. The following amplification conditions were used: an initial denaturation step of 94 ◦ C for 3 min was followed by 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 58 ◦ C for 20 s, and extension at 72 ◦ C for 50 s, and a final extension at 72 ◦ C for 7 min. PCR products were analyzed on a 1% agarose gel before screening for heterozygosity by single strand conformational polymorphism (SSCP) as described previously (Curchoe et al., 2005). Briefly, denatured PCR products were resolved on a 12% polyacrylamide gel at 4 ◦ C for 20 h. The polyacrylamide gel was then silver-stained (Bosari et al., 1995), dried and archived. The presence of two bands confirmed the heterozygosity of the samples. 2.3. Allele-specific expression of the imprinted cattle IGF2R gene For all samples identified as heterozygous for the IGF2R SNP, one step RT-PCR was performed using the one-step RT-PCR enzyme mix (Qiagen). RNA was reverse transcribed using the following conditions: 50 ◦ C for 30 min and 95 ◦ C for 15 min before PCR amplification using the conditions described above. The RT-PCR products were then analyzed for allelic expression by SSCP as described above. 2.4. Quantitative real-time RT-PCR of cattle IGF2R Total RNAs (20 ng) from tissues collected at day 25, day 45 and day 75 of gestation were reversetranscribed at 50 ◦ C for 1 h with Superscript III (Invitrogen). The real time RT-PCR reaction mixture contained 5 l of cDNA, 12.5 l of SYBR green master mix (Applied Biosystems Inc., CA, USA) and 0.3 m of forward and reverse primers in a total volume of 25 l. All tissues were analyzed in triplicate for every gene. The specificity of the real time RT-PCR product was proven by a melting curve analysis. To compare the relative amounts of gene expression in different samples, the comparative cycle (CT) method, also known as the 2−CT method, was applied as we described previously, using the following formula: CT = CTsample − CTcalibrator , where CT = threshold cycle for the target gene
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amplification, CT = CTtarget gene − CTendogenous reference . The endogenous reference gene chosen for this study was the ˇ-actin gene. The calibrator was a mixture of RNA from liver and lung tissues and was included in each real time amplification reaction. All validation and quantification were conducted as suggested by the manufacturer of the real time amplification kit (Applied Biosystems Inc.). Validation of the amplification efficiency of target genes and the endogenous reference gene was completed before using the 2−CT method for quantification. Briefly, different dilutions of cDNA (range tested) were amplified by real time RT-PCR using the gene specific primers (IGF2R, 5 -CCGGGAGATGGTAATGAGCA-3 (forward) and 5 -TCTCGTTCTCGTCGGCCT-3 (reverse) and -actin, 5 -ACCGTGAGAAGATGACCCAGA-3 (forward) and 5 -TCACCGGAGTCCATCACGAT-3 (reverse)). The corresponding CT values were plotted against the log of each cDNA amount and the data were plotted using linear regression analysis. The values of the slopes of the validation curves for each gene was less than 0.1, indicating that the same amplification efficiency was obtained for all samples with both greater and lesser amounts of the cDNA. 2.5. Expression of the bovine IGF2R antisense transcript, AIR RT-PCR primers were designed to amplify an intronic region between IGF2R exons 1 and 2, thereby ensuring that only the IGF2R antisense transcript, AIR would be amplified. The sequences of the forward and reverse primers were 5 -AGAGCGTGGCTGAGTCTGACG-3 and 5 -CGAGACCCCACCAGACTAGAC-3 . Total RNA was amplified with the Qiagen One-Step RT-PCR kit with 1000 ng of total RNA, 0.6 pmol/l primers and 2 l reverse transcriptase in a total volume of 25 l at 55 ◦ C for 30 min. 2.6. Statistical analysis Differences in the relative amounts of mRNA of IGF2R in different organs between cloned and control animals were analyzed using the Kruskal–Wallis subroutine (a non-parametric alternative to one-way independent sample ANOVA) of the Statistical Analysis System (SAS) and a probability value of p ≤ 0.05 was considered to be significant. 3. Results 3.1. Genotyping for bovine IGF2R and presumptive parental allele determination All day 25, day 45, day 75, aborted, dead and live clones and age-matched control cattle were confirmed for heterozygosity of an SNP in the 3’UTR of the IGF2R gene. A specific band of 298 bp was amplified and sequenced. A G/A transition was identified at position 52 (Fig. 1(A)). The heterozygous and homozygous patterns are revealed by SSCP as two and one band for each sample (Fig. 1(B)). The G allele was preferentially expressed in the fibroblast and cumulus cells of the donor Holstein–Friesian dairy cow used to create the day 25, day 45 and day 75 clones (Yang et al., 2005) (data not shown). The A allele was preferentially expressed in the donor cells of the beef animal used to create the aborted, living and cloned animals that died shortly after birth (Fig. 1(B)). Due to the lack of parental tissues for the donor animals, the parental origin of the G and A alleles could not be determined. However, based on the fact that the maternal allele is the expressed allele in naturally reproduced cattle, it was assumed that the G and A alleles are the maternal alleles in Holstein and beef cattle donor animals, respectively. 3.2. Allele-specific expression of the imprinted IGF2R gene in clones and controls In control animals, one allelic expression pattern of IGF2R was observed in all tissues, including extra-embryonic tissues at day 25 (Table 1) and day 45 (Table 1) of gestation, organ tissues collected at day 75 of gestation (Table 2) and from newborn calves (Table 3). This gene expression pattern was consistent with predominate expression of the presumptive maternal allele as indicated by the darker band with a lesser amount of “leaky” expression of the presumptive imprinted/paternal allele, as indicated by the lighter band (Fig. 1(B)). The only exception to this finding was observed in the brains
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Fig. 1. (A) Sequence chromatogram of PCR product shows the SNP, a G to A transition, at the bp 52 position (arrow) of the cattle IGF2R gene. (B) SSCP genotyping of the SNP in the 3’UTR of the cattle IGF2R gene. Lanes 1–3, represent a heterozygous (G/A) sample and homozygous (AA) and (GG) samples, respectively. Lanes 4–9, demonstrate different patterns derived from all samples; Lane 4, the heterozygous (AG) pattern; Lane 5, allelic expression of the donor cells used to create the clones in Table 1, showing mostly allele A expression with leakage of the G allele; the A allele is assumed to be the maternal allele. Lanes 6 and 8, display examples of homozygous GG allelic expression, Lanes 7 and 9 display examples of homozygous AA allelic expression.
of newborn controls, were the IGF2R gene was bi-allelically expressed, i.e. the two bands representing the expression of the maternal and paternal alleles were equal in intensity (Fig. 1(B)). Because this is the first detailed characterization of allelic expression in conventionally bred cattle, the “leaky” imprinting pattern is assumed to be the normal pattern of IGF2R gene expression in cattle in all tissues with the exception of the newborn brain, which is bi-allelic.
Table 1 Allelic expression patterns of the IGF2R gene in the trophectoderm of day 25 and day 45 controls and clones. Capital letters represent the parental allele with the strongest expression (M = maternal, P = Paternal). Extra-embryonic tissues
Day 25
Day 45
Controls Clone #1 Clone #2 Clone #3 Clone #4
pM ” ” ” ”
pM ” ” ” P
Symbol (”) indicates no difference in allelic expression from the control. Table 2 Allelic expression patterns of the IGF2R gene in organs/tissues of day 75 control and cloned fetuses. Capital letters represent the parental allele with the strongest expression (M = maternal, P = Paternal). Day 75
Controls Clone #1 Clone #2 Clone #3 Clone #4
Heart
Brain
Liver
Spleen
Cotyledon
Intercotyledon
pM ” ” ” ”
pM ” ” ” ”
pM ” ” ” ”
pM ” P PM PM
pM ” ” ” ”
pM ” ” ” ”
Symbol (”) indicates no difference in allelic expression from controls.
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Table 3 Allelic expression patterns of the IGF2R gene in living clones, clones which died shortly after birth (approximately 280 day), aborted clones, near-term fetuses (approximately 250 day) and fetuses derived from conventional breeding. Capital letters represent the parental allele with the strongest expression (M = maternal, P = Paternal). Approximate Day 250–280
Controls Living #1 Living #2 Living #3-6 Aborted #1 Aborted #2 Aborted #3 Dead #1 Dead #2 Dead #3 Dead #4
Brain
Cotyledon
Heart
Kidney
Liver
Lung
Placenta
MP
Mp MP MP ” ” P P ” ” ” MP
Mp
Mp
Mp
Mp
P P MP ” ” MP MP
mP P MP ” ” MP mP
P P M ” ” ” mP
MP P MP ” ” mP mP
Mp ” ” ” P M P ” ” ” ”
P M ” ” ” ”
Symbol (”) indicates no difference in allelic expression from controls. Blank areas indicate that tissues were not collected.
In cloned fetuses, the normal “leaky” imprinting pattern of IGF2R in cattle was observed in all extraembryonic tissues collected at day 25, and 3 out of 4 extra-embryonic tissues collected at day 45 of gestation. One of the day 45 extra-embryonic tissue displayed complete monoallelic gene expression without “leakage” (Table 1). However, the expressed allele was the putative paternal allele which normally only displays “leaky” gene expression. While most organs of day 75 cloned fetuses had normal expression patterns, Clone 1 was also switched to exclusive expression of the paternal allele in the spleen. In Clones 3 and 4, the spleen also displayed the abnormal biallelic expression pattern of IGF2R (Table 2). The most variable allelic expression patterns, out of all the age groups studied, were observed in spontaneously aborted cloned calves. Every tissue tested could display exclusive switching (no “leakage”) to the paternal allele (Table 3) in different cloned animals. Only one instance of normal allelic gene expression was observed in this group (in the cotyledon of aborted clone 1). Several abnormal instances of exclusive maternal expression, skewing to the paternal allele and equal expression of both alleles were also observed in this group. Two cases of skewing toward the paternal allele were observed in the group of clones that died shortly after birth, one in the kidney, liver and lung of one calf and one in the lung of another (Table 3). The brains and placentas of clones that died shortly after birth and the placentas of living clones all displayed the same allelic expression as those of the controls. Representative SSCP gels are shown in Fig. 2. 3.3. Quantitation of IGF2R expression Amounts of IGF2R gene expression were measured in all tissues of day 75 controls and cloned animals (Fig. 3) and in the trophectoderm of day 25 and day 45 cloned and control animals. In fetuses produced by conventional reproduction, the spleen is the major producer of IGF2R, while the liver, lung, kidney, and placenta produce intermediate amounts of IGF2R. In samples from the present study, the brain and heart produced negligible amounts of IGF2R. This pattern of tissue-specificity was maintained in cloned animals. Interestingly, the expression of the IGF2R gene in all of the tissues was similar for cloned fetuses and control fetuses with the exception of the spleen, for which the cloned fetuses had lesser amounts when compared to control fetuses. Despite the lack of significant differences between controls and clones in the steady state amounts of IGF2R gene expression in most tissues, individual clones displayed more or less in a number of tissues, as evidenced by the large standard error bars for the clones (Fig. 3). For instance, day 75, clone 3 displayed 26 and 11% of IGF2R gene expression (relative to controls) in the spleen and lung, respectively, while showing 462% more than controls in the liver. In one instance (day 75, clone 2) IGF2R gene expression was consistently less, displaying 55,
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Fig. 2. Representative SSCP gels of the allelic expression of IGF2R in organs/tissues of living clones, clones which died shortly after birth, aborted clones and at day 25,day 45 and day 75 of gestation. (A) Lanes 1–4: representative gel of alleles AG, AA, GG and the donor cell pattern, respectively, Lanes 5–8: representative of the leaky pattern seen in the heart, liver, lung and spleen of newborn control fetuses. (B) Lanes1–3: representative image of normal “leaky” gene expression observed in the heart, kidney and liver of a cloned calf that died shortly after birth, Lanes 4 and 5: representative image of the normal biallelic expression seen in the brain of cloned cattle that died shortly after birth. Lanes 6–8: representative image of the switched mono-allelic expression seen in the heart, kidney and liver of an aborted cloned calf. The data for all cloned animals are summarized in Table 1.
27, 8 and 1% expression of IGF2R in the spleen, cotyledon, lung and brain, respectively, compared to the average amounts in the corresponding tissues in controls. The trophectoderm of day 25 and day 45 cloned fetuses showed no significant difference in amounts of gene expression when compared to controls of the same age group. The placentas of the living clones showed normal “leaky” expression of IGF2R.
Fig. 3. Relative amounts of IGF2R gene expression as folds over the calibrator for organs/tissues of day 75 cloned (solid bar) and control (open bar) animals. The symbol (*) represents a significant difference of p < 0.05.
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Fig. 4. Representative agarose gel image AIR expression in all tissues tested in the organs/tissues of control and cloned cattle at day 75 of gestation. Lane 1: 1 kb + ladder, Lane 2: negative control, Lanes 3–8: brain, cotyledon, kidney, liver, lung, inter cotyledon of a control animal, Lanes 8–11: inter cotyledon, lung and liver of a cloned animal.
3.4. Expression of the IGF2R antisense transcript, AIR All control and cloned organ/tissue samples expressed AIR as detected by RT-PCR (Fig. 4). No difference was observed in AIR expression in tissues expressing different IGF2R alleles. 4. Discussion In the present study, the IGF2R allelic expression pattern and amounts of gene expression throughout development of the placenta, were established by examining day 25 trophectoderm (before implantation), 45 trophectoderm (during implantation) and 75 placenta and cotyledons (after implantation), and in live healthy term cattle by SSCP and real time RT-PCR. Allelic expression was found to be monoallelic with “leakage” of the imprinted allele in day 25, day 45, day 75 and term cattle placental structures. Age-matched placental structures from clones were compared to controls and no differences were found in the relative amounts of IGF2R gene expression between day 25 and day 45 cloned and control trophectoderm using the Kruskal–Wallis subroutine of the SAS. When comparing the rate of change between the day 25 and day 45 trophectoderm to that of the control group, no differences were found. While no overall differences were found in the cotyledon and intercotyledon of day 75 fetuses between clones and controls, individual clones had a large amount of variation, such as (day 75) clone 2, which had 60% of IGF2R gene expression in the cotyledon compared to the control group. These data are consistent with the results of Lonergan et al. (2007) whose data have suggested that large calf syndrome manifests after day 45 of gestation. The allelic expression pattern of IGF2R in the cotyledon and intercotyledon area of healthy, live cloned cattle was consistent with the data from the control cotyledon and inter cotyledon area, with most allelic expression patterns showing monoallelic expression with “leaky” expression of the imprinted allele. In sharp contrast to this the placentas and cotyledons of spontaneously aborted cattle displayed abnormal monoallelic expression of the maternal allele or allele switching to the paternal allele. Normal “leaky” expression of the IGF2R gene was not observed in this group of samples. IGF2R allelic expression patterns and amounts of gene expression in the organs of day 75 cloned and control fetuses were highly variable in some individual clones compared to age matched controls. No differences in amounts of IGF2R gene expression were found in most organs, when taken together as a group, and the allelic expression data correlates well with this observation. Most day 75 organ samples display monoallelic expression with “leakage”, which compares well with the data from Yang et al. (2005). However, the expression of the IGF2R gene in the spleen was found to be different when compared to the control group, which correlates well with the allelic expression data observed in the present study. Two instances of monoallelic, paternally switched expression were observed and most other samples appeared biallelic, instead of displaying the typical “leaky” expression pattern. Organs from cloned calves that died shortly after birth display normal “leaky” gene expression patterns, similar to age matched controls. Organs from spontaneously aborted fetuses, however, had the greatest variability in IGF2R gene expression patterns. Every organ tested was capable of display-
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ing abnormal allelic expression in different animals. Only one case of normal allelic expression was observed in this entire group (the cotyledon of aborted clone #1). The observed allelic switching was not due to a lack of AIR expression because AIR was detected in all tissues examined, including those in which allele switching occurred. Normally, AIR is expressed from the paternal allele, and it silences the expression of the paternal IGF2R by interfering with the initiation of IGF2R’s paternal promoter. It is unclear if, in the tissues that expressed the paternal allele of IGF2R, AIR is also switched to the maternal allele because we did not have a polymorphism to investigate this possibility. Further studies can explore the regulation of gene expression in tissues displaying allele switching. Because less expression of the IGF2R gene in one organ does not predict less expression in other organs (with the notable exception of day 75 clone #2), it is possible that an epigenetic abnormality may have occurred early in development before the onset of organogenesis, as suggested by Smith et al. (2005). Taking these data together, proper allelic expression of IGF2R has a role in terms of sustaining a typical period of gestation, as evidenced by the group of aborted fetuses, which displayed the greatest allelic irregularities, in contrast to the group of day 75 cloned fetuses, which displayed the least allelic irregularities. It is concluded that proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Acknowledgements The authors would like to thank Hamed Kian and Yuqin Zhang for control tissue collection from the slaughterhouse. 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