Correlation of Developmental Differences of Nuclear Transfer Embryos Cells to the Methylation Profiles of Nuclear Transfer Donor Cells in Swine

CORRELATION OF DEVELOPMENTAL DIFFERENCES OFNUCLEAR TRANSFER EMBRYOS CELLS TO THE METHYLATIONPROFILES OF NUCLEAR TRANSFER DONOR CELLS IN SWINE

Aaron J. Bonk1, Hee-Tae Cheong1,2, Rongfeng Li1, Liangxue Lai1, Yanhong Hao1, ZhonghuaLiu1, Melissa Samuel1, Emily A. Fergason1, Kristin M. Whitworth1, Clifton N. Murphy1, EricAntoniou1, and Randall S. Prather11 Division of Animal Science, University of Missouri-Columbia, Columbia, MO 65211

2 School of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, Korea

AbstractMethylation of DNA is the most commonly studied epigenetic mechanism of developmentalcompetence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNTprocedures have examined the effects of different culture media on donor cells and reconstructedembryos, and the methylation status of specific genes in the fetus or live offspring. Here we used amicroarray based approach to identify the methylation profiles of SCNT donor cells including threeclonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney andmammary tissues. The methylation profiles of the donor cells were then analyzed with respect totheir ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2,A7, and A8 had blastocyst rates of 11.7%a, 16.7%ab, and 20.0%b, respectively (ab P<0.05). Adultsomatic cells included kidney, mammary (large), and mammary (small) also had different blastocystrates (ab P<0.05) of 4.2% a, 10.7% ab, and 18.3% b, respectively. For clonal donor cells and for adultsomatic cell groups the donor cells with the highest blastocyst rates also had methylation profileswith the lowest similarity to the methylation profiles of the in vivo-produced blastocysts. Conversely,the donor cells with the lowest blastocyst rates had methylation profiles with the highest similarityto the methylation profiles of the in vivo-produced blastocysts. Our findings show there is an inversecorrelation to the similarity of the methylation profiles of the donor cells and the in vivo-producedembryos, and to the blastocyst rates following SCNT.

KeywordsGlobal DNA Methylation; Embryo; Somatic Cell Nuclear Transfer; Swine; Embryo Development

INTRODUCTIONSomatic cell nuclear transfer (SCNT) has successfully produced live animals in manymammalian species including mice, pigs, sheep, goats, rats, cats, dogs, and bovine. Many cellstypes have been used as donor cells to produce live, fertile offspring. The donor cells could beclassified as either: 1) blastomeres from the early embryo, 2) embryonic stem cells (ES), 3)somatic stem cells, or 4) fully differentiated somatic cells. The use of blastomeres as donor

E125 Animal Science Research Center, 920 East Campus Drive, 573-882-6414, FAX 573-884-7827, [email protected] Conflicts of InterestThe authors are not aware of any conflicts of interest.

NIH Public AccessAuthor ManuscriptEpigenetics. Author manuscript; available in PMC 2008 September 1.

Published in final edited form as:Epigenetics. 2007 September ; 2(3): 179–186.

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cells were successfully used to generate the first mammalian clones 1–3, Embryonic stem cellshave been successfully used as donor cells in the mouse 4, 5. Higher rates of development havebeen reported when using ES cells as the donor cells compared to somatic donor cells 6, 7.Conversely, the use of inbred 129 ES cells failed to produce any offspring that survived morethan 1 day 8. Since true ES cells have not been isolated from mammals other than the mouse,cultured somatic cells are the most common source of the donor karyoplasts in SCNT.

Adult somatic cells were first used as donor cells to produce the sheep, Dolly 9. Live offspringhave been produced by using donor cells from a wide variety of sources including fetalfibroblasts 7, 9–11, adult fibroblasts 12, 13, and somatic cumulus cells 14, 15. Cloned micewere even produced by using natural killer T (NKT) cells 16. NKT cells were shown to havethe same developmental potential as adult fibroblasts and cumulus cells 17 but greater thanlymphocytes 18. In general, the use of fetal and adult somatic cells in nuclear transfer isextremely inefficient in producing live offspring. These studies support the theory that thedevelopmental potential is inversely correlated to the differentiation status of the donor cells19. The highest developmental potential was observed when the donor nuclei are taken fromthe zygote and any additional development results in decreased developmental potential,presumably through a mechanism that increases resistance to epigenetic remodeling.

Previous studies have examined the methylation status and the expression of imprinted genesin the embryos and offspring derived by using SCNT. The specific question we were interestedin was to determine if SCNT donor cells that have a high percent of development to theblastocyst stage following nuclear transfer have methylation profiles that are more similar tothe methylation profile of in vivo-produced blastocysts as compared to those that result in lowerrates of development to the blastocyst stage. Our hypothesis is that there will be greatersimilarity between the methylation profiles of donor cells with high developmental potentialand in vivo-produced blastocysts than between the methylation profiles of donor cells with lowdevelopmental potential and in vivo-produced blastocysts.

In this study, we examined the developmental potential of cultured clonal cells derived froma primary preparation of porcine fetal cells and of donor cells selected from kidney andmammary cells that were not cultured prior to SCNT. The methylation profiles of these donorcells were determined by using Porcine Differential Methylation Hybridization (PDMH)microarrays 20. The methylation profiles were then correlated to the developmental potentialof the cells.

MATERIALS AND METHODSOocyte Procurement and In Vitro Maturation

Cumulus-oocyte-complexes (COCs) were aspirated from ovaries from prepubertal gilts thatwere collected from a local abattoir. Germinal vesicle stage oocytes were either collected forPDMH analysis or matured in vitro prior to in vitro fertilization. The COCs were incubated inTissue Culture Medium 199 (Gibco BRL, Grand Islands, NY) containing 0.1% (w/v) PVA, 10ng/ml (w/v) epidermal growth Factor, 0.57 mM cysteine, 0.5 μg/ml (w/v) porcine folliclestimulating hormone and 0.5 μg/ml (w/v) porcine lutenizing hormone 21. The maturationmedium was pre-equilibrated in 5% CO2 in air at 39ºC overnight. COCs were matured for 40–44 hours in 5% CO2 at 39ºC prior to the removal of the cumulus cells by vortexing for threeminutes in Hepes-buffered medium with 0.1% (w/v) hyaluronidase. Denuded oocytes werewashed 3X and held in modified Tris-buffered medium 22 prior to fertilization or SCNT.

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Nuclear Transfer Embryo ProductionReconstructed embryos were produced using SCNT techniques as previously described 23.Porcine fetal fibroblast-like (PFF) cultures were established from a day 35 porcine fetus. Afterthe second passage the cells were plated in to 96-well plates one cell to each well and culturedin Dulbecco’s Modified Eagle Medium (DMEM) containing 15% (v/v) fetal calf serum (FCS),75 μg/ml (w/v) penicillin G, and 50 μg/ml (w/v) streptomycin (Pen-Strep) in 5% CO2 at 39ºC.PFF colonies were harvested and transferred to 4-cell culture dishes (Nunc, Rochester, NY)for about 26–28 population doublings prior to freezing in DMEM/15% (v/v) FBS/10% (v/v)DMSO. Three clonal cell sub-lines were used as donor cells in SCNT (Fig. 1A). Sevenreplications were conducted with each of the donor cell lines used on the same day, therebyallowing direct comparison of the data.

Kidney and mammary tissues were collected from two full term sows and single cellsuspensions were produced from the tissues after treatment with collagenase for 8 hours (Fig.1B and Fig. 1C). The cells were frozen in 90% (v/v) FCS and 10% (v/v) DMSO. Small, roundcells with smooth membranes were collected from the kidney cell preparation. Small roundcells with smooth membranes and large cells with rough, asymmetrical membranes wereselected from the mammary cell preparation. Six replications for mammary-small (MS) andmammary-large (ML) cells and five replications for kidney cells (K) were conducted on thesame day thereby allowing direct comparison of the data.

Kidney and Mammary Donor Cell CultureThe kidney and mammary donor cells were selected and transferred to culture medium in orderto characterize these in vitro growth and morphological characteristics. Donor cells (n=150)from kidney and mammary tissues were selected and cultured under oil in 200 μl DMEM/20%(v/v) FCS/Pen-Strep in 5% CO2 at 37ºC for 7 days. As a positive control, the original single-cell preparations were left in culture medium and cultured along with the selected donor cells.This procedure was repeated twice.

Animal CareAll procedures within were reviewed and approved by the University of Missouri InstitutionalAnimal Care and Use Committee, and were performed in accordance with the GuidingPrinciples for the Care and Use of Laboratory Animals.

Statistical AnalysisTreatment means and cell number data were analyzed with the SAS General Linear ModelsProcedure (SAS Institute, Inc., Cary, NC) by using Duncan’s multiple range test.

Porcine Differential Methylation HybridizationPorcine DMH was conducted as previously described 20. Porcine CpG island clones from aPorcine CpG Island Library (PCGIL) (United Kingdom Human Genome Mapping Project,Hinxton, Cambridge, United Kingdom) were cultured in 96 well plates. The cloned insertswere amplified by polymerase chain reaction (PCR) using the library specific primers. PCRproducts were stored at −20ºC until needed. Restriction digestion with Bstu I was performedandthe digested and undigested PCR products were run on a 1.5% 0.5X TBE agarose gel. BstuI positive clones where the PCR product was cut indicating the presence of a Bstu I site (CGCG)in the insert, were reracked and recultured in 96 well plates. Plates with Bstu I positive cloneswere PCR amplified preparation for printing. The purified PCR products were dried andresuspended in 10 μl 50% DMSO/1% CHAPS 24.

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The resuspended PCR products were printed on Gold Seal glass microscope slides (FisherScientific, Hampton, NH) that were coated with 0.02% (w/v) poly-L-lysine (Sigma, St. Louis,MO) in 0.5X PBS 25. The slides were stored for 3 weeks at room temperature under desiccationbefore printing with a pick and place robot. The printed slides were cross linked at 120 mJ/cm2 for 20s (Spectrolinker; Spectronics Corp., Westbury, NY) prior to blocking in 0.018% (w/v) succinic anhydride (Sigma, St. Louis, MO) and 0.043 M sodium borate (Sigma, St. Louis,MO) in 1-methyl-2-pyrrolidinone (Sigma) 25. The slides were stored under desiccation and atroom temperature until hybridization.

DNA IsolationThe DNA was isolated from the donor cells by adding H2O to a final volume of 25 μl andincubating at 98ºC for 15 minutes.

Amplicon Generation, Labeling and HybridizationAmplicons were produced by digesting the donor cell DNA with the restriction enzyme MseI (50 units) in 1X NEB 2, and 1X BSA at 37ºC overnight as recommended by the supplier(NEB). The restricted DNA was ligated to PCR linkers produced by mixing oligomers (H-24,5′-AGG CAA CTG TGC TAT CCG AGG GAT and H-12, 5′-TAA TCC CTC GGA), heatingto 65ºC, and cooling to room temperature. The DNA was digested with the methylationsensitive restriction enzyme Bstu I (NEB) as recommended. The intact DNA fragments wereamplified by PCR using H-24 as the linker specific primer. The PCR program consisted of adenaturation step at 98ºC for 5 minutes followed by 40 cycles of denaturation at 95ºC for 1minute, annealing at 55ºC for 1 minute and extension for 72ºC for 1 minute. A final extensionof 72ºC for 10 minutes completed the program.

The PCR products were labeled with amino allyl-dUTP using the BioPrime labeling systemas previously described 20 with modifications. The PCR products were purified andresuspended in 29 μl H2O, mixed with 1X Bioprime buffer, dNTPs, Klenow, and incubatedfor 60 minutes at 37ºC. Amino allyl-dUTP incorporated PCR products were purified andthesamples were dried and resuspended in 0.1 M sodium carbonate buffer (pH 9.0) and mixedwith Cy3 for the donor cell DNA or mixed with Cy5 for the liver reference sample. The sampleswere incubated for 60 minutes at room temperature. The labeling reactions were purified withQiaquick columns. and the labeling efficiency was then analyzed spectrophotometrically byusing a Nanodrop ND-1000 (Nanodrop, Wilmington, DE). Comparable amounts of labeledtest sample and liver reference sample, based on the incorporation of the Cy 3 and Cy5 dyes,were mixed together. The combined samples were purified, dried, and resuspended inhybridization buffer. The samples were denatured at 95ºC for 3 minutes and immediatelytransferred to ice before being applied to a microarray slide with a lifterslip (Erie Scientific,Portsmouth, NH). The microarrays were hybridized at 42ºC for 8–12 hours before removingthe lifterslip in Wash I (1X SSC/0.2% (w/v) SDS), and washing in Wash II (1X SSC/0.2% (w/v) SDS), Wash III (0.1X SSC/0.2% (w/v) SDS), Wash IV (0.1X SSC), and Wash V (H2O).The slides were immediately dried by using centrifugation at 1,500×g for 5 minutes, andscanned with an Axon 4000B scanner (Molecular Devices Corp. (MDC) Sunnyvale, CA).

Microarray AnalysisMicroarray images were initially analyzed with GenePix 4.0 (MDC) and spots with intensitieswhere at least 25% of the pixels were greater than 1 standard deviation from the backgroundin either the Cy3 or Cy5 channel were further analyzed with Gene Spring version 7.2 (AgilentTechnologies, Santa Clara, CA). The LOWESS normalized data was analyzed by ANOVA(P<0.05) assuming all variances to be equal, using the Benjamini and Hochberg FalseDiscovery Rate for multiple testing. Specific clones were selected for sequencing based on thesimilarity or significant difference in the methylation profiles of donor cells and in vivo-

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produced blastocysts. Donor cell methylation profiles and bisulfite sequencing results werecompared to the liver and in vivo-produced blastocyst methylation profiles and bisulfitesequencing results previously described 20.

Bisulfite Sequencing AnalysisThe DNA from the clonal donor cells was treated with bisulfite by using the EZ DNAMethylation-Gold Kit (Zymo Research, Orange, CA) according to the vendor’srecommendations. Primers (Table S1) were designed for bisulfite treated DNA by using theMethPrimer software 26. PCR was performed with the following components (H2O 32.5 μl,DNTP 1.3 μl, 10X Buffer (TagGold) 5 μl, MgCl2 5 μl, Forward Primer (10 μM) 2 μl, ReversePrimer (10 μM) 2 μl, DNA (Bisulfite treated) 2 μl, AmpliTaq Gold (5 u/μl) 0.25 μl for a totalof 50 μl. The PCR program consisted of a denaturation step at 98ºC for 3 minutes followed by50 cycles of denaturation at 95ºC for 15 seconds, annealing at 55ºC for 30 seconds andextension for 72ºC for 30 seconds. A final extension of 72ºC for 5 minutes completed theprogram.

The PCR was purified by using the Qiaquick columns and were cloned by using the pGEM T-Easy Kit (Promega, Madison, WI). The vectors were transformed in to DH10B cells(Invitrogen, Carlsbad, CA) and grown on LB/IPTG/X-Gal/Ampicillin agar plates.Recombinant colonies were selected for sequencing based on the blue/white screening criteria.The cytosines of the CpG sites were identified as methylated or unmethylated if a C or T waspresent in the sequence, respectively. The percent methylation was calculated for the respectivesequence and the methylation status of the microarrays and bisulfite sequencing werecompared. A ratio of liver:donor cell methylation was calculated by using the followingformula: Rm = (100 − MS)/(100 − ML); where: MS is the average % CpG methylation for asequence in the sample, and ML is the average % CpG methylation for a sequence in the liverreference.

The use of this formula provides a means to calculate a ratio that indicates the relative levelsof methylation in a given sequence when one of the samples lacks methylated CpGdinucleotides. The ratios produced from the microarray and bisulfite analysis were classifiedas consistent when the bisulfite analysis-produced ratio indicated the sample washypomethylated (>1) or hypermethylated (<1) and matched the hypermethylation status of themicroarray-produced data. From the microarray-produced ratios, the samples were classifiedas hypermethylated when the ratio was <0.75 and the sample was classified as hypomethylatedwhen the ratio was >1.25.

RESULTSDevelopment of Reconstructed Embryos after Somatic Cell Nuclear Transfer

The donor cells were initially characterized by determining the blastocyst rate following SCNT.The percent blastocyst for the clonal cell lines were significantly higher (p<0.05) for A8(20.0%) than for A2 (11.7%) (Table 1A). Significant differences were not observed for thefusion rates, cleavage rates or the mean cell numbers for the clonal cell lines. The percentblastocyst for the MS donor cells (18.3%) was significantly higher (P<0.05) than the K (4.2%)donor cells (Table 1B). The cleavage rate was significantly higher (P<0.05) for the ML andMS donor cells (46.7% and 45.9%) than for the K donor cells (26.2%). Mean cell numbers ofthe blastocysts for the K, MS, and ML cells were 22.8, 23.6, and 26.3, respectively. Significantdifferences were not observed for the fusion rates or the mean cell numbers for the ML, MS,and K donor cells. These results demonstrate that there are the donor cells have significantdifferences in directing development after SCNT.

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Differential Methylation in Donor Cells and Blastocyst Stage EmbryosGlobal methylation analysis of the donor cells and the blastocyst stage embryos was performedby using the custom-made PMDH microarrays. Microarray data was analyzed by using theGeneSpring 7.2 software to perform an ANOVA assuming all variances to be equal, P<0.01using the Benjamini and Hochberg False Discovery Rate for multiple testing. Of the 2,445clones that were analyzed, 380 (15.5%) were found to be significantly different in at least oneof the biological conditions of donor cells and in vivo-produced blastocysts. Identification ofthese differentially methylated regions provides regions of interest for additional analysis.

A condition tree was generated based on the methylation profiles of the clonal cell lines, kidneycells, mammary cells and the in vivo-produced blastocysts by using the GeneSpring 7.2software (Fig. 2). Samples with high levels of similarity in the overall methylation profiles aregrouped together in the condition tree. Increasing disparity in the methylation profiles resultsin the samples being located farther apart in the condition tree. Conversely, the donor cellswith the highest blastocysts rates after SCNT [A7 (16.7%), MS (18.3%), and A8 (20.0%)]grouped away from the in vivo-produced blastocysts. These results show that the similaritiesof the donor cell and in vivo-produced blastocyst methylation profiles appear to be inverselycorrelated with the developmental potential of the donor cells after SCNT.

Bootstrap analysis by using the TIGR Multiple Array Viewer software was used to validatethe condition tree generated by using the GeneSpring 7.2 software (Fig. S1: supplementalinformation can be found at http://animalsciences.missouri.edu/faculty/prather/).Unfortunately, the TIGR Multiple Array Viewer software used to do the bootstrap analysisdoes not include the same correlation analysis that is used by the GeneSpring software.Specifically, the Standard Correlation used in the GeneSpring software is commonly referredto as Pearson correlation around zero. The TIGR Multiple Array Viewer does not contain thiscorrelation procedure so the Pearson Correlation analysis was substituted. Therefore, cautionshould be used in attempting to extrapolate the bootstrapping results to the clustering generatedby using GeneSpring. The strongest support was provided for the clustering of the MS and invivo-blastocysts. Furthermore, A7 and A8 were the most different cells from the in vivoproduced blastocysts, confirming the analysis with GeneSpring.

Donor Cell CultureDonor cells from the kidney and mammary tissues were characterized by identifying the growthcharacteristics in DMEM/20% FCS/Pen-Strep. After 7 days in culture, no cells were observedin the culture drops for the K or the MS cells. Conversely, cells were observed to be growingfrom the ML donor cell group. These cells had a spindle shape associated with fibroblast-likecells or a “cobblestone” shape associated with endothelial cells. These differences in growthcharacteristics suggest there are particular media requirments for the specific cells types.

Bisulfite Sequencing AnalysisSpots previously identified as differentially methylated in the donor cells and the blastocystgroups were selected for additional analysis by using bisulfite modified PCR 20. Themethylation status for 5 bacterial clones (B G2, HH A7, K D3, S E3, and X G2) was determinedin the liver or the clonal cell lines A2 or A7 (Fig. S1). The methylation liver/donor cellmethylation ratios were calculated for 5 clones using DNA from the clonal cell lines A2 andA7 and compared to the microarray results (Table S2). The PDMH values are LOWESSnormalized Cy5/Cy3 ratios representing the methylation status of the specified clones in theliver (Cy5) and in the clonal donor cells (Cy3) samples. The Bisulfite Analysis values representthe relative methylation levels in the liver and in the clonal donor cells at selected regions ofthe specified clones. The Bisulfite Analysis values were calculated from the equation shownin the Materials and Methods section. Bisulfite Analysis data and the microarray analysis data

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are in agreement for all of the samples. Differential methylation hybridization microarrayanalysis of the methylation levels of the donor cells was validated by the bisulfite modificationPCR analysis and depicted graphically (Fig. S3).

Similarity of Donor Cells and In Vivo-Produced Blastocyst Methylation ProfilesThe similarity of methylation profiles in the donor cells and the in vivo-produced blastocystswere analyzed by using Self Organizing Map analysis. Clones that had similar hypomethylationand hypermethylation to in vivo-produced blastocysts clustered together and are shown inFigure 3. The complete Self Organizing Map analysis is presented in Figure S2, and the BLASTanalysis of the sequenced genes is shown in Table S3. The Cy5/Cy3 ratios of the normalizedratios are shown where the reference sample was labeled with Cy5 and the test sample waslabeled with Cy3. The Cy5/Cy3 ratio of one indicates equivalent methylation levels. A Cy5/Cy3 ratio greater than one indicates there is less methylation in the test sample than in thereference sample. Conversely, a Cy5/Cy3 ratio less than one indicate that the test sample ismore methylated than the reference sample. Of particular interest are those groups that showa general trend that correlates to the blastocyst rate. These results suggest that the genesassociated with the clones in these groupings may be important in the regulation ofdevelopmentally relevant genes. Alternatively, these clones may represent regions that areresistant to epigenetic remodeling during early development and this resistance results in lowerdevelopment rates following SCNT.

DISCUSSIONTo date, studies of epigenetics and SCNT typically involved the identification of factors relatedto culture conditions that affect the methylation of the donor cells and the reconstructedembryos, or to the identification of errors in epigenomic reprogramming at some point afterthe SCNT procedure. The objective of this study was to correlate the methylation profiles ofvarious donor cells prior to SCNT to the developmental potential of the respectivereconstructed embryos following SCNT. Our hypothesis was that the donor cells with thehighest blastocyst rate, measured by the blastocyst rate after in vitro culture, would havemethylation profiles that were the most similar to the methylation profiles of in vivo-producedblastocysts. Similar to previous work 27 the results presented here also identify subpopulationsof somatic cells in tissues that have differential potential to direct the development ofreconstructed embryos. This differential potential to direct development occurs with donorcells that have undergone extended culture as well as with donor cells that have not beencultured. The significantly lower cleavage rate and blastocyst rate observed in the kidney donorcells imply an intrinsic resistance of the epigenome to reprogramming following SCNT.

We used a microarray based approach to characterize the methylation profiles of the donorcells from adult somatic tissues and fetal tissues and the methylation profiles of in vivo-produced blastocysts. Donor cells from kidney tissues were found to have methylation profileswith the highest similarity to in vivo-produced embryos and the lowest blastocyst rate followingSCNT of all the donor cells. Conversely, the methylation profiles of the small mammary cellsand the clonal cell lines A7 and A8 were found to be the most dissimilar to the in vivo-producedblastocyst, yet these donor cells yielded the highest rate of blastocyst development. It shouldbe noted that the entire blastocyst, including the inner cell mass and the trophoblast cells, wasincluded in generating the methylation profile. The effect of analyzing this mixed populationof cell types is not known since the differences in the global methylation status of the in vivoinner cell mass and trophectoderm has not been extensively studied.

One reason mouse ES cells have been used in SCNT is because it is thought that the epigeneticprogramming is limited in such a way that there will be minimal reprogramming needed tomimic that of the early blastomere thereby resulting in higher rates of development. Somatic

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cells are used as donor cells in all other animals since true ES cells have yet to be identified.Here we showed that the lowest rates of blastocyst development were shown to be associatedwith cells that were the most similar to the methylation profile of the in vivo-producedblastocyst.

The donor cells used in the current study were either clonally derived or were selected, basedon morphology, from kidney or mammary cells that were not cultured in vitro. The goal ofboth strategies was to start with a homogenous population of donor cells thereby minimizingthe likelihood that only a small, select population of donor cells in a primary culture are mainlyresponsible for the development of the reconstructed embryos. Cells were identified in theclonally derived cells and also the uncultured cells that resulted in high, medium, and low ratesof blastocyst development. These results support the idea that successful nuclear transferrequires reprogramming of the donor cells. This basic idea had been intuitively acceptedwithout the presence of a comprehensive study that would control for the technical problemsthat may affect development. Hiragi and Solter 28 recently demonstrated that thedevelopmental rate of reconstructed embryos is inversely correlated to the stage of the embryosfrom which the donor blastomeres were collected. These results indirectly supports otherstudies where the rates of blastocyst development and the production of live offspring followingSCNT decreases when donor cells are collected from progressively later stages of the earlymouse embryo 28, 29 and the early cattle embryo 30.

Most nuclear transfer experiments have used donor cells that were cultured in vitro. In thisexperiment, we used adult somatic cells that had not been extensively cultured in vitro. Thesegroups were included to assess the effect of minimizing the effect that culture medium andextended culture times have on the methylation status and blastocyst rates following SCNT.The large disparity we observed in the blastocyst rates of the kidney cells and the smallmammary cells was not expected. This difference in developmental potential seems to indicatethat the epigenetic status of the selected kidney cells is significantly more resistant toreprogramming and remodeling than the other donor cells.

The inability of the kidney cells and the small mammary cells to grow in standard culturemedium suggests that there is a selection process when primary cultures are established fromfetal or somatic tissues. These cells may require specific media that contains growth factorssuch as LIF, EGF, PDGFA, FGF2, ECGF1, and insulin 31. The presence of cells in the adultmammary tissues that result in high blastocyst rates following SCNT, but are likely excludedfrom most primary cell culture preparations, presents a potential source of a stem cell-like cellpopulation that is highly abundant in mammary tissues and possibly most other tissues.Optimization of the selection procedure and of the culture media for these cells could create areadily available population of cells with the developmental potential that is functionallysimilar to isogenic embryonic stem cells. Potentially homologous subpopulations ofmultipotent cells in the adult mouse testis have been found to contribute to the multiple organsafter injection in to the early blastocyst 32. In vitro culture in the appropriate media resultedin the development of these cells to derivatives of the three germ layers.

DNA methylation is the most common form of epigenetic modification but additional factorsare known to affect gene regulation in an epigenetic manner. Histone modifications such asmethylation, acetylation, and phosphorylation are known to regulate expression and to beheritable to daughter cells. Aberrant DNA methylation associated with cancer is oftenassociated with chromatin modifications such as acetylation of H3 33 and H4 34. Also, thePolycomb/Trithorax group response elements are associated with various histonemodifications including methylation and ubiquitination and are capable of function in anepigenetic manner in up-regulating and down-regulating expression (Reviewed by Ringroseand Paro 35). In addition, RNA has also been implicated as an epigenetic factor whereby

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processes including chromatin remodeling, transcription factor binding, and transcription areaffected by RNA signaling (Reviewed by Mattick 36. Further studies of donor cells epigeneticcontributions would benefit from the inclusion of histone modification and regulatory RNA-related effects on developmental competence.

The two groups of donor cells could be contrasted by the age of the cells. The kidney andmammary cells were derived from an adult female animal whereas the clonal cells were derivedfrom a day-30 fetus. While DNA methylation can change in normal tissue as an age-relatedprocess 37 these potential differences do not appear to have played a specific role with thedevelopmental potential of reconstructed embryos. Again, the two groups of donor cells, adultsomatic cells (kidney and mammary) and the clonally-derived fibroblast-like cells, had groupsthat ranged from low to high developmental potential suggesting that the age of the cells andthe age-related changes in DNA methylation was not a critical factor in the developmentalpotential of the cells.

In conclusion, this study shows that a wide range of developmental potential is present in donorcells regardless of whether the cells were in extended in vitro culture. While the focus herewas to better understand epigenetics caused by DNA methylation, other epigenetic marks arelikely present on the chromatin that regulates nuclear reprogramming. The similarity of themethylation profiles of the donor cells to the in vivo-produced blastocyst shows an inversecorrelation to blastocyst rate following nuclear transfer. Therefore, the epigenetic condition ofsome donor cells is resistant to the detrimental effects of extended culture on donor cells, andthere are subpopulations in somatic cells that show variable resistance to epigeneticremodeling, in this case DNA methylation, following SCNT.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

Acknowledgements

The authors would like to acknowledge surgical skills of Tom Cantley and David Wax; August Rieke for gatheringsamples and supplying animals; and Nathan Bivens of the University of Missouri-Columbia DNA core for his expertassistance with the DNA sequencing. Funding for this project was from the National Institutes of Health the NationalCenter for Research Resources (R01 RR013438) and Food for the 21st Century. Supplemental figures and tables canbe found at http://animalsciences.missouri.edu/faculty/prather/.

AbbreviationsA2

A7, A8, Designation for three different donor cell populations

ANOVA analysis of variance

BSA bovine serum albumin

COC cumulus-oocyte complex

DMEM Dulbelcco’s modified eagle medium

DMSO dimethyl sulfoxide

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ES embryonic stem cell

FCS fetal calf serum

K kidney cells

ML mammary cells- large

MS mammary cells- small

NEB New England Biolabs

NKT natural killer cells

PCGIL porcine CpG island library

PCR polymerase chain reaction

PDMH porcine differential methylation hybridization

PFF porcine fetal fibroblast

PVA polyvinyl alcohol

SCNT somatic cell nuclear transfer

SDS sodium didocylsulfate

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Figure 1.A. Clonal cell lines cultured (A2, A7 & A8) and trypsinized (A2′, A7′ & A8′).B. Somatic donor cells from mammary (A) and kidney (B) tissues. Arrows indicate the large(L) and small (S) cells that were selected from the mammary cells.

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Figure 2.Hierarchical clustering of the methylation profiles of the clonal donor cells (A2, A7, and A8,),somatic cells (kidney, (K), Mammary-Large (ML), and Mammary-Small (MS)), and in vivo-produced blastocysts. Developmental potential is negatively correlated to similarity to the invivo-produced blastocyst methylation profile. Donor cells that with the lowest blastocyst ratesafter SCNT had the most similar methylation profiles while donor cells with higher blastocystrate did not cluster with the in vivo-produced blastocysts. The blastocyst rate after SCNT isshown in parentheses below each of the donor cell types.

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Figure 3.Clones with similar methylation profiles in the donor cells and the in vivo-produced blastocystswere clustered by using Self Organizing Map analysis. Hypermethylation (A) andhypomethylation (B) of the donor cells are correlated with lower blastocyst rates after SCNT.Those shown above were found to be different (P<0.01) in at least one of the biological samplesof donor cells.

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Table 1In vitro development of SCNT-produced embryos derived from porcine donor cells. A. The blastocyst rates for theclonal cell lines A2, A7, and A8 were significantly different (a,b in the same column, P<0.05) after SCNT. A significantdifference was not observed for the fusion rate, cleavage rate, or blastocyst cell number. B. The blastocyst rates for thedonor cells Kidney, Mammary-Large, and Mammary-Small were significantly different (a,b in the same column,P<0.05) after SCNT. A significant difference was not observed for the fusion rate, cleavage rate, or blastocyst cellnumber.

ACell-line No. (%) of fused/

manipulatedNo. (%) of cleaved No. (%) of blastocyst # Cell (mean±SE) in blastocysts

(range)A2 94/139 (67.6) 66 (70.2) 11 (11.7)a 25.5±2.0 (17–42)A7 84/133 (63.2) 63 (75.0) 14 (16.7)ab 24.3±1.6 (18–40)A8 95/131 (72.5) 67 (70.5) 19 (20.0)b 25.7±1.4 (19–37)BCell-line No. (%) of fused/

manipulatedNo. (%) of cleaved No. (%) of blastocyst # Cell (mean±SE) in blastocysts

(range)Kidney 103/224 (46.0) 27 (26.2)a 4 (4.2)a 22.5± 2.2 (16–27)Mammary-Large 77/179 (37.5) 35 (46.7)b 8 (10.7)ab 23.6± 1.3 (21–25Mammary-Small 77/179 (43.0) 34 (45.9)b 22 (18.3)b 26.5±2.6 (19–44)


Correlation of Developmental Differences of Nuclear Transfer Embryos Cells to the Methylation Profiles of Nuclear Transfer Donor Cells in Swine

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