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Gene expression profiling of individual bovine nuclear transfer blastocysts

¹ AgResearch Ruakura, Hamilton, New Zealand and ² University of Waikato, Department of Biological Sciences, Hamilton, New Zealand

Correspondence should be addressed to P L Pfeffer; Email: peter.pfeffer{at}agresearch.co.nz

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. In this study we focused on the switching on of embryonic genes by screening with a microarray consisting of 5000 independent cDNA isolates derived from a bovine blastocyst library which we constructed for this purpose. Expression profiling was performed using linearly amplified RNA from individual day 7 nuclear transfer (NT) and genetically half-identical in vitro produced (IVP) blastocysts. We identified 92 genes expressed at lower levels in NT embryos whereas transcripts of 43 genes were more abundant in NT embryos (P 0.05, 1.5-fold change). A range of functional categories was represented among the identified genes, with a preponderance of constitutively expressed genes required for the maintenance of basal cellular function. Using a stringent quantitative SYBR-green real time RT-PCR based approach we found, when comparing the means of the expression levels of a larger set of individual embryos, that differences were small (< 2-fold) and only significant for two of the seven analysed genes (KRT18, SLC16A1). Notably, examination of transcript levels of a single gene in individual embryos could not distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Mammalian nuclear transfer (NT) cloning has produced live, viable offspring in many species, yet cloning efficiencies are always very low, with generally less than 4% of reconstructs reaching term (Wilmut et al. 2002). Early gestational losses of NTembryosare often associated with aberrant placental development in livestock and mice (Hill et al. 2000, De Sousa et al. 2001, Ogura et al. 2002). Anomalies in foetal and perinatal stage cloned calves and lambs are heterogeneous, including excessive birth weight (large offspring syndrome), defects in the gastrointestinal, cardiopulmonary, hepatic and renal systems as well as skeletal malformations (Wells et al. 1997, Zakhartchenko et al. 1999, 2001, Lanza et al. 2001, Gibbons et al. 2002, Pace et al. 2002, Rhind et al. 2003). Furthermore, survival beyond term is also affected with annual mortalities of 8% reported in cattle (Wells et al. 2004) and early death in mice (Ogonuki et al. 2002).

The primary cause of defects in clones is postulated to be epigenetic, based on the observation that abnormalities of clones are not transmitted to subsequent generations (Shimozawa et al. 2002, Tamashiro et al. 2002, Wells et al. 2004). For the survival of a NT reconstructed embryo, the epigenetic memory of the donor cell nucleus has to be erased and the chromatin remodeled into an embryo-equivalent state in a reprogramming process that leads to the correct initiation of the embryonic gene expression programme. Such chromatin remodelling in clones is often incomplete as demonstrated by aberrant DNA methylation patterns in bovine NT embryos (Bourc’his et al. 2001, Dean et al. 2001, Kang et al. 2001, 2002). Likewise, histone lysine methylation and acetylation changes in bovine clones showed disparity relative to control embryos (Santos et al. 2003). Observations that NT mouse embryos, in contrast to control embryos, developed more efficiently in donor cell culture medium than in embryo culture medium, implies that donor cell transcription may not have ceased after transfer (Gao et al. 2003). Improved viability was observed when using embryonic stem (ES) as opposed to somatic cells (Hochedlinger & Jaenisch, 2002). ES cells more closely resemble embryos in terms of their gene expression profile than do differentiated cells and hence less nuclear reprogramming may be required upon using ES cells for NT. Thus the superior NT efficiencies when using ES cells suggests that correct embryonic genome activation constitutes a major hurdle during somatic nuclear transfer.

Gene expression studies in nuclear transfer embryos have been published for several species. In mice, pluripotency genes involved in early development have been reported to be misregulated in cloned embryos (Boiani et al. 2002, Bortvin et al. 2003). The expression of cattle NT blastocysts has been previously studied in an attempt to identify marker genes that would predict clone developmental competence (Daniels et al. 2000, Donnison & Pfeffer 2004). Many of the previous studies measured the expression of only a small number of genes by semi-quantitative RT-PCR (Daniels et al. 2000, 2001, Wrenzycki et al. 2001, Park et al. 2003). Detailed quantitative analyses of individual blastocysts has revealed a large degree of embryo to embryo variation and only subtle changes in expression levels (Camargo et al. 2005). These studies, however, offer only a limited picture of the gene transcription changes occurring after nuclear transfer. In order to obtain a more complete picture of the accuracy and extent of nuclear reprogramming, a global method is required. One group has recently used such an approach, creating a microarray from cDNA derived from cultured genital ridge cells, and identified 18 genes as significantly differentially expressed between NT and in vitro produced (IVP) embryos (Pfister-Genskow et al. 2005).

We have here addressed this issue further, focusing on gene activation in NT embryos. To this end we have constructed a novel bovine blastocyst cDNA library and synthesised a 5000 feature blastocyst-stage microarray, allowing us to compare embryonic gene expression in individual NT blastocysts with that of genetically half-identical IVP blastocysts. Genes identified from the microarray as differentially expressed were analysed further by quantitative real time RT-PCR. We interpret our results in terms of a stochastic model for reprogramming.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
IVP and NT embryo production
Abattoir recovered ovaries were aspirated, with oocytes matured in vitro and used to generate either zona-free IVP or NT blastocysts. Generation of IVP embryos by in vitro fertilisation (IVF; using sperm from bull AESF 1) was as previously described (Thompson et al. 2000) with the exceptions of zona removal after IVF and single embryo culture. Bovine NT, using cultured skin fibro-blasts recovered from bull AESF 1 and embryo culturing of both the IVPand NTembryos using a synthetic oviduct fluid system, with embryos cultured singly has been previously described (Oback et al. 2003). Grading and staging of development according to published guidelines (Robertson & Nelson 1998) was performed by only one of us (D.N.W.). Briefly, grade 1 embryos were symmetrical with well-defined and uniform blastomeres. Grade 2 embryos had moderate irregularities in the shape or size of the inner cell mass or similar irregularities in the size, colour or density of the individual blastomeres. Single embryos were washed in PBS and transferred in a minimal volume into individual tubes. These were immediately flash frozen in liquid nitrogen and kept at – 80 ° C until RNA extraction. A reference standard was created from a pool of 200 zona-intact day 7 IVP blastocysts.

RNA isolation and amplification
For the amplification procedure, RNA was isolated from individual embryos and the reference standard pool using the RNAqueous micro kit (Ambion, Austin, TX). The manufacturer’s protocol was followed with the following modifications. To each thawed sample 200 ng of poly-deoxy-inosinic-deoxy-cytidylic acid (poly[d(I-C)]; Roche, Mannheim, Germany) was added. The elution volume of 40 µl was reduced to 10 µl with a SVC100H speedvac concentrator and the DNase I treatment step was omitted. Non-stick microfuge tubes were used (Neptune #3435.S3, Raylab, Auckland, New Zealand).

Individual blastocyst samples were subjected to two rounds of linear amplification using the Arcturus RiboAmp RNA amplification kit (Arcturus, Gene Works, Auckland, New Zealand) using a modified version A. The modifications included using the centrifuge conditions described in version C of the manufacturer’s protocol and a first round in vitro transcription length of 5 h at 42 ° C and 4 ° C overnight. For the second round of in vitro transcription, reagent volumes were doubled and samples incubated for 6 h at 42 ° C then 4 ° C overnight. Sample yields and the integrity of the amplified antisense RNA (aRNA) was examined spectrophotometrically and by gel electrophoresis. Yields were as follows (in µg): IVP embryos—35, 35, 53 and 71; NT embryos—67, 81, 33, 34 and 25. aRNA was stored at – 80 ° C.

For the reference standard, the first round of linear amplification was performed with the Arcturus kit and the yield determined by spectrophotometer. Aliquots (280 ng) were made to which 200 ng poly[d(I-C)] was added. For the second linear amplifications a modified version of the protocol by Wang and colleagues was used (Wang et al. 2000). Modifications included the use of Microcon YM-100 columns (Millipore, North Ryde, Australia) for double-stranded DNA clean-up, an incubation period of 5 h at 37 ° C and 4 ° C overnight for the in vitro transcription reaction and the use of the RNAqueous micro kit (Ambion) for aRNA clean-up. All second round amplifications of the standard reference were pooled and the yield quantified with a spectrophotometer. The standard reference aRNA was stored at – 80 ° C.

cDNA blastocyst library construction
Total RNA was obtained from 640 day 7 and 8 bovine IVP blastocysts using the Trizol procedure (Invitrogen, Auckland, New Zealand). PolyA⁺ RNA was isolated using the MicroPolyA Purist kit (Ambion). PolyA⁺ RNA (160 ng was reverse-transcribed following the SMART cDNA protocol (PT3000-1, 2001; Clontech, BD Biosciences, Auckland, New Zealand), but using SuperscriptII (Invitrogen) for reverse transcription. Protocol PT3000-1 was followed using 24 cycles for LD-PCR amplification. cDNA ligated to TriplEx2 vector was packaged using MaxPlax Packaging Extract (Epicentre, Madison, WI) and titered using E. coli XL-1 blue cells according to standard protocols.

Microarray generation
The primary pTriplEx2 library was plated at low density on 15 cm plates containing IPTG and X-Gal to allow for individual recombinant white plaques to be picked. Five thousand plaques were randomly selected and each added to 50 µl -dilution buffer (100 mM NaCl, 10 mM MgSO₄, 35 mM Tris–HCl pH 7.5, 0.01% gelatine) and 2 µl of chloroform in 96-well plates, before storage at 4 ° C. The insert of each phage was amplified in a 50 µl PCR reaction containing 2 µl phage, 0.25 µl each of primers TriplEx5' (5 '-CTCGGGAAGCGCGCCATTGTGTTGGT-3' ) and 3' (5'-ATACGACTCACTATAGGGCGAATTGGCC-3' ; Clontech), 5 µl 10 x PCR buffer (Roche), 1 µl 10 mM dNTP and 0.2 µl 5 Units/µl Taq DNA polymerase (Roche). Cycling conditions were 94 ° C for 5 min, followed by 40 cycles of 94 ° C for 15 s, 58 ° C for 30 s and 72 ° C for 2 min, with a final extension of 72 ° C for 7 min. Two microlitres were used for gel electrophoresis to examine the amplified inserts.

Preparation of microarray slides, preparation of PCR -amplified DNA for microarray printing and microarray printing was according to published protocol (Diez-Tascón et al. 2005). The microarrays were printed with an ESI array robot (Toronto, Canada) using a 32 pin head. Each pin head printed a block of 180 spots. We included 243 blank spots (no DNA) per array. Array details can be accessed at the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under A-MEXP-312.

cDNA labelling and microarray hybridisation
Two micrograms of amplified aRNA from the individual blastocyst samples and aliquots of the reference sample were converted into cDNA and aminoallyl-labelled using the Invitrogen Superscript cDNA Labelling System according to the manufacturer’s protocol. Single embryo samples were labelled with Cyanin (Cy) 5 dye and the standard reference sample with Cy3 (Amersham, Auckland, New Zealand). After the final elution the two samples to be compared on each microarray were combined, 10 µl 3 M (pH 5.3) sodium acetate and 2 µl 20 mg/ml glycogen (Roche) added before ethanol precipitation and resuspension in 10 µl DEPC-water. Microarray pre-hybridisation, hybridisation and post-hybridisation conditions were as previously described (Diez-Tascón et al. 2005). Dye-reversal hybridisation where the standard reference was labelled with either Cy3 or Cy5 and the two samples hybridised against one slide gave a correlation coefficient of 0.995.

Microarray analysis
Microarray slides were scanned at a resolution of 10 µm by a GenePix 4000A microarray scanner (Axon Molecular Devices, Sunnyvale CA). Cy 5 and Cy 3 fluorescence was measured at a 16 bit pixel resolution. Tagged image files were converted into data files using GenePix Pro 4.1 software (Axon). GenePix analysis included automatic as well as manual flagging of bad spots. Microarray data was manipulated and displayed using Data Desk 6.1 (Data Description, Ithaca, NY) and deposited in the public ArrayExpress database under accession number E-MEXP-556. Graphical displays were also generated using S-PLUS 6.1 (Insightful Corporation, Seattle, WA) and Microsoft Excel. Statistical analysis was conducted using a spatial mixed model (Baird et al. 2004) with GenStat software (VSN International, Oxford, UK). Differential expression between the mean of the IVP and NT blastocyst samples was determined using a t-test, with an arbitrary cut-off of 1.5-fold change and a 5% significance level. Reproducibility in hybridisation was monitored by including 384 features spotted in duplicate at different positions within each microarray slide. Comparison of the log intensity ratios across this data set revealed an average correlation ration of 0.83.

Sequencing of significantly differentially expressed probes
Inserts of interest were PCR amplified as described above and sequenced (Eck et al. 2004) from the 5' direction using the TriplEx5' LD primer (#9107-1; Clontech) before separation on a Prism 3100 DNA sequencer (Applied Biosystems). Sequence information was analysed using BLASTN searches of public databases. An expected value of e^{– 10} or less was considered to be a significant match.

Total RNA isolation, DNase treatment and reverse transcription
Single day 7 zona-free IVP or NT blastocysts were placed in 100 µl of Trizol (Invitrogen, Auckland, New Zealand) to which 5 pg rabbit -globin mRNA (Sigma, Sydney, Australia) and 800 ng of MS2 RNA (Roche, Auckland, New Zealand) were added. Samples were extracted with 20 µl of chloroform followed by the addition of 10 µg linear acrylamide (Ambion, Austin, Texas) and 65 µl of cold isoproanol. After 10 min at room temperature, samples were centrifuged at 14 000 rpm (16 000 g) for 30 min, washed with 150 µl 70% ethanol and air-dried. After resuspension in 7 µl DEPC-treated water, 2 µl 1 U/µl RNase-free DNase1 (Invitrogen, Auckland, NZ) and 1 µl of 10 x DNase I buffer was added and samples incubated for 1 h at 37 ° C. Samples were precipitated with 1.5 µl 3M sodium acetate (pH 5.5) and 45 µl 100% ethanol, washed in 70% ethanol and resuspended in 12 µl DEPC-treated water.

To each sample 1 µl 10 mM dNTP and 1 µl 10 mM oligo dT₁₄VN anchored primer (Invitrogen) were added before incubation at 65 ° C for 5 min. Four microlitres 5 x first strand buffer (Invitrogen), 1 µl 40 U/µl Protector RNase inhibitor (Roche), 1 µl 200 U/µl Superscript III (Invitrogen) were added and the samples incubated at 50 ° C for 60 min, then 70 ° C for 15 min. A reverse transcription negative (RT–) control was included. This was followed by the addition of 0.5 µl 2 U/µl RNAse H (Invitrogen) for 30 min at 37 ° C. After the addition of 2 µl sodium actetate (pH 5.5) samples were passed through Qiaquick mini elute columns (Qiagen, Auckland, New Zealand) and resuspended in 40 µl of TE buffer in non-stick 0.65 ml tubes (Neptune #3435.S3, Raylab, Auckland, New Zealand).

Real time PCR analysis
Seven of the genes identified by the microarray as differentially expressed—cytochrome c oxidase I; keratin 18 (KRT18); myosin, light peptide 6, alkali, smooth muscle and non-muscle (MYL6); ribosomal protein L21 (RPL21); solute carrier family 16, member A1 (SLC16A1); ß-tubulin isoform 5(TUBB); and tyrosine 5-monooxygenase/tryptophan 5-monooxygenase activation protein, theta isoform (YWHAQ)—were selected for quantitative expression analysis by SYBR-green real time PCR. In addition, glyceraldehyde-3-phospate dehydrogenase (GAPDH) and keratin 8 (KRT8) were examined. Primers (Table 1) were designed using GCG’s Prime (Accelrys, San Diego, CA) and, where possible, were selected to cover putative intron sequences as determined by comparison to the homologous human gene loci.

Standard curves were obtained using PCR fragments that were excised from a 1% agarose gel, purified using a Roche Gel Extraction Kit, resuspended in TE and quantified with both a NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE) and fluorometrically with PicoGreen (Invitrogen) using the LightCycler. Standards consisted of a 10-fold dilution series containing 10⁵ to 10 copies/µl. Sample concentrations calculated from the standard curves were converted into an estimate of copy number per blastocyst after correcting for recovery and reverse transcription losses using values obtained for -globin recovery. The average measurement for each blastocyst sample (at least two sample values) was used in statistical analysis of real time PCR results by an unbalanced ANOVA using GenStat (VSN).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Experimental strategy
We here endeavoured to obtain a global picture of the effect of NT on the expression of genes transcribed at the blastocyst stage. To this end we made a novel bovine blastocyst cDNA library allowing the synthesis of a blastocyst-specific microarray. Our bovine day 7 blastocyst cDNA library had a complexity of 1.1 million. The use of oligo-dT mediated reverse transcription ensured inclusion of the 3' trailers of cDNA isolates, a necessary requisite when hybridising to linearly amplified cDNA which exhibits a similar 3' bias. Blue/white selection on X-Gal plates showed over 90% of phages to contain inserts. Amplification of inserts by PCR using vector specific primers yielded an insert size ranging from 0.1 to 5 kbp with a median of 0.7 kbp. For the generation of our blastocyst-specific microarray, 5000 insert-containing clones were randomly selected from the primary non-amplified library, PCR amplified and spotted.

A microarray approach necessitated an amplification step to generate sufficient cDNA to hybridise to the arrays, in particular as we wished to examine the expression profile of individual blastocysts, in line with observations that each NT reconstructed embryo exhibits a unique expression profile (CS, DNW, PLP, unpublished observations). We used linear mRNA amplification to achieve this. To assess the degree of bias introduced by this technique, total RNA isolated from two pooled IVP blastocysts was divided equally and both samples concurrently subjected to two rounds of amplification. Comparison of the amplified cDNA after hybridisation to our microarray (Fig. 1A) revealed a strong correlation (r = 0.99) between the separate amplifications (Fig. 1B). We conclude that minimal bias was introduced by the linear amplification procedure, yet a small number of false positives are unavoidable.

View larger version (15K): [in this window] [in a new window]   Figure 1 Testing bias resulting from linear amplification. (A) Experimental design for linear amplification bias control testing. RNA derived from 2 blastocysts was split equally and linearly amplified independently (A and B). (B) Plot of the fluorescence intensity generated by hybridising Cy5 labelled amplification A and Cy3 labelled amplification B (hyb 1) target to the 5000 features of the blastocyst-specific microarray. Note the dye-reversal microarray comparison (Hybridisation 1R) also displayed a correlation coefficient of r = 0.99 (not shown).

Microarray results
Statistical analysis using a spatial mixed model (Baird et al. 2004) identified 164 microarray features (spots) as demonstrating significant differential expression between the means of the NT and IVP blastocysts (P 0.05, 1.5-fold change). Thus of the 5000 features examined, 3% were consistently differentially expressed in the NT blastocysts. Of these features, 121 demonstrated lower expression and 43 higher expression in NT relative to IVP blastocysts. A graphical representation of the mean-adjusted relative expression intensities of each individual blastocyst across the 164 selected features highlights feature variation in individual embryos but also reveals the consistency of differences between the groups of IVP and NT embryos (Fig. 2). In particular, genes normally expressed at the blastocyst stage tend to be expressed at lower levels in NT embryos.

View larger version (22K): [in this window] [in a new window]   Figure 2 Heat map of the mean adjusted relative expression intensity (log2[IVP/NT]) of the microarray identified differentially expressed genes (P 0.05, 1.5-fold change). Columns represent individual blastocyst samples, rows represent individual genes. White lines represent absence of a microarray value.

View this table: [in this window] [in a new window]   Table 2 List of genes identified from the microarray analysis as significantly differentially expressed between NT and IVP blastocysts (P 0.05, 1.5-fold change)a.

View larger version (24K): [in this window] [in a new window]   Figure 3 Classification of microarray identified significantly differentially expressed genes into functional categories, with each category expressed as a percentage of the total. Within each category, the fraction of genes which are underexpressed in NT embryos are shaded, those overexpressed are not.

View larger version (23K): [in this window] [in a new window]   Figure 4 Individual and average gene expression levels (in thousands of transcripts per embryo) in IVP and NT day 7 blastocysts as determined by quantitative real time RT-PCR. Expression in individual blastocysts is represented by diamonds (IVP, n = 16; NT, n = 17) with the mean and standard deviation (back transformed from log scale) depicted adjacent to each data set. Each individual blastocyst sample was measured twice, once at a 1:2 dilution. Cyt C; cytochrome C oxidase I; KRT, cytokeratin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The stochastic nature of the gene expression profile differences in individual NT embryos
During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. Incomplete reprogramming has been proposed to underlie the loss of viability that is known to occur in NT-derived embryos. In our hands and using skin fibroblast donor cells, development to term and beyond of transferred NT embryos is less than 10% compared to around 40% for IVP blastocysts (Kruip & den Daas 1997, Oback et al. 2003, Smeaton et al. 2003, Wells et al. 2003, 2004).

An interesting question regarding incomplete reprogramming concerns the issue of whether particular gene loci are preferentially affected (reprogramming error hotspots) or whether defects are of a stochastic/random global nature. Consistent gene expression changes at a defined set of loci would support the hotspot scenario whereas unpredictability of expression profiles would suggest a stochastic model of reprogramming. Expression profile studies using pools of embryos (Wrenzycki et al. 2001, Donnison & Pfeffer 2004) are unsuitable for answering this question as stochastic effects are averaged out. Studies of individual NT-generated foetuses or newborns (Humpherys et al. 2001) are severely biased as these animals had already survived many critical phases of embryogenesis during which a large fraction of NT embryos succumb. We thus compared the gene expression of individual NT and genetically half-identical IVP embryos at an early stage (blastocyst) using a global microarray approach. We concentrated on the switching on of embryonic genes by screening a microarray consisting of cDNA derived from a blastocyst library which we constructed for this purpose. Genetic and developmental variation as well as embryo culture effects were minimised in this study.

We identified 92 genes expressed at lower and 43 genes expressed at higher levels in NT embryos (P< 0.05, 1.5-fold change). The preponderance of underexpressed genes in NT embryos when examining a set of cDNA isolates known to be expressed at the blastocyst stage suggests an impaired capacity of NT embryos for activation of embryo-specific genes. However, a range of functional categories was represented among the identified genes, with a high proportion involved in protein biosynthesis, signalling, cytoskeleton, mitochondrial, protein binding/folding and metabolism/biosynthesis. Most of these genes are not embryo-specific but would be expected to be constitutively active in every cell as they are required for the maintenance of the basal cellular machinery. Misregulation of these genes in NT embryos therefore points to errors that may not be directly attributable to the donor cell’s transcription profile.

It should be pointed out that our results are derived from examining a subset of blastocyst-specific genes. Currently it is believed that about 10 000 genes are transcribed in the mammalian blastocyst (Zeng et al. 2004). Based on frequency distributions of expressed sequence tags (ESTs) in non-normalised cDNA libraries (in-house data), we estimate our 5000 randomly chosen microarray features to represent around 1700 different genes. Of these genes we observed 8% to be consistently (P< 0.05) over- or underexpressed by at least 1.5-fold (none of sample deviated by more than 2.6-fold). Based on this random sample of around 1700 genes the 95% confidence interval for misexpression of genes after nuclear transfer lies in the range of 6.7–9.4%.

Interestingly, our quantitative data indicated that only a proportion of genes found to be misexpressed in the microarray data set are consistently misregulated when examining a larger set of embryos. On comparing the means of the expression levels of these misregulated genes, the changes seen are small (< 2-fold). Considering the range of variation detected in the IVP samples, examination of transcript levels of a single gene in individual embryos cannot distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.

Studies in the mouse can also be interpreted in light of stochastic reprogramming. Thus the expression of Oct4 was found to be independent to that of a transgenic Oct4-enhancer driven reporter gene in individual NT derived blastocysts (Boiani et al. 2002). In a non-quantitative study of 10 putative pluripotency genes in mouse NT embryos, the expression profile was found to differ from embryo to embryo (Bortvin et al. 2003). In the bovine, detailed measurements of transcript levels of a small set of genes in individual embryos has yielded no (Camargo et al. 2005) or only one (CS, DNW, PLP, unpublished data) candidate that is significantly different in a set of IVP and NT embryos. Yet for that candidate, as for GAPDH, KRT18 and SLC16A1 identified in this study, changes in levels are small and, as in the mouse, seen only for a subset of NT embryos. Lastly, the stochastic reprogramming error model would easily account for the observed heterogeneity in the timing and cause of nuclear transfer associated defects: the manifestation of reprogramming errors would be dependent on the particular functions of the random set of genes affected.

Genes expressed at lower levels in NT embryos
The genes we found here to be more frequently misexpressed in NT embryos were KRT18, SLC16A1 and GAPDH. GAPDH may represent genes whose regulation is influenced by multiple regulatory pathways as it codes for an enzyme implicated in a wide range of cellular processes including glycolysis, tRNA binding, cytoskeleton interaction by the binding of tubulin and microtubules, apoptosis, vesicular transport and telomere length regulation (Sirover 1999, Tisdale 2002, Sundararaj et al. 2004). GAPDH is often used for gene expression normalisation (Bustin 2000, Pfister-Genskow et al. 2005). Our finding illustrates the need for caution in the choice of genes used for normalisation. Exogenous standards circumvent this problem.

The membrane transporter SLC16A1 regulates the uptake of monocarboxylates including pyruvate (Garcia et al. 1994). Pyruvate uptake increases twofold after the expanded blastocyst stage in IVP embryos, suggesting a metabolic requirement for pyruvate at this stage (Rieger et al. 1992). Thus a reduced expression of SLC16A1 in NT embryos may affect their viability. Interestingly our microarray identified reduced mRNA levels for three additional membrane solute carrier transporters, suggesting an altered metabolism in NT blastocysts.

The cytoskeletal keratin proteins KRT18 and KRT8 have been previously detected at early embryonic stages (Chisholm & Houliston 1987). In the only other global study examining gene expression in individual NT preimplantation embryos using micro-arrays, KRT8 was found to be underexpressed in clones (Pfister-Genskow et al. 2005). We examined KRT8 expression in our NT embryos using stringent criteria for quantification but found no significant difference in the mean expression levels between our NT and genetically half-identical IVP blastocysts. Though this discrepancy may have arisen from the use of different protocols for NT production and gene quantification in the two studies, it may also simply reflect the stochastic nature of reprogramming errors.

The biological relevance of the multiple small changes in expression is not clear, although it has previously been shown that an increase or decrease of only 50% in Oct4 expression induced alternative differentiation pathways in mouse embryonic stem cells (Niwa et al. 2000). As every NT embryo appears to be quite unique in terms of its transcript levels, future studies will have to attempt to correlate gene expression profiles of individual embryos with their subsequent development, a technically daunting task.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We thank Dr Peter Eck for valuable advice on sequencing and Sue Beaumont, Andria Green, Pavla Misica, Fleur Oback, Jan Oliver, Anita Schurmann, Hilda Troskie as well as Drs Debbie Berg and Björn Oback for help with the production of embryos. This work was supported by the New Zealand Foundation for Research, Science and Technology. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 16 September 2005
First decision 21 October 2005
Revised manuscript received 26 October 2005
Accepted 27 February 2006

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