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Refereed/Peer-reviewed Amongst a range of changes that have taken place within tertiary education, perhaps the most revolutionary has been a shift to student-centred approaches focused on life-long learning. Accompanying this approach to holistic higher education (HE) has been a growing interest in, and understanding of, the Scholarship of Teaching and Learning (SoTL). SoTL has, at its core, a deep concern with student learning and is therefore well-aligned with higher education’s renewed focus on its students. In this conceptual paper, we examine the impact of the T-shaped person which many tertiary institutions are operationalizing to inform and connect the development of students’ deep disciplinary knowledge with non-academic and employment readiness skills (such as communication, problem-solving, teamwork, and critical thinking). Importantly, we argue for a re-positioning of SoTL to complement and support this model, with SoTL as both the fulcrum and the fluid, multiple threads of discourse that are intricately entwined around the structure of the T-shaped model. We encourage our colleagues to strive to be T-shaped practitioners and we cast a vision of a T-shaped community. Here, all stakeholders within HE connect both their academic knowledge and holistic skills in collaborative ways to produce learners who flourish in modern society. The SoTL community plays a pivotal role in achieving this vision and is well-positioned to expand the current notion of SoTL toward a more holistic, interconnected, central role in HE.

Space exploration has long been a source of inspiration, challenging scientists and engineers to find innovative solutions to various problems. One of the current focuses in space exploration is to send humans to Mars. However, the challenge of transporting materials to Mars and the need for waste management processes are two major obstacles for these long-duration missions.To address these two challenges a process called Astroplastic was developed that produces polyhydroxybutyrate (PHB) from solid human waste, which can be used to 3D print useful items for astronauts. PHB granules are naturally produced by bacteria such asRalstonia eutrophaandPseudomonas aeruginosafor carbon and energy storage. ThephaJ, phaC,andphaCBAgenes were cloned from these native PHB-producing bacteria intoEscherichia coli. These genes code for enzymes that aid in PHB production by converting products of glycolysis and β-oxidation pathways, such as acetyl-CoA and enoyl-CoA, into PHB. To ensure a continuous PHB production system and to eliminate the need for cell lysis to extract PHB, recombinant E. coli was engineered to use the genes in its natural type I secretion system to secrete PHB. The C-terminal of the HlyA secretion tag was fused to phasin (PhaP), a protein originally fromR. eutropha. Phasin-HlyA electrostatically binds PHB granules and transports them outside of the cell.In addition to genetically engineering bacteria, a concept for start-to-finish PHB production process was designed. Integrating expert feedback and experimental results, conditions for each step of the process including the collection and storage of waste, volatile fatty acid (VFA) fermentation, VFA extraction, PHB fermentation, and PHB extraction were optimized. The optimized system will provide a sustainable and continuous PHB production system, which will address the problems of transportation costs and waste management for future space missions.Financial DisclosureMindfuel Science Alberta Foundation Genome Alberta GenScript Polyferm Canada GeekStarter Alberta Integrated DNA Technologies University of Calgary University of Calgary Cumming School of Medicine University of Calgary Bachelor of Sciences University of Calgary Schulich School of Engineering University of Calgary O’Brien Centre for the Bachelor of Health Sciences City of Calgary Alberta Innovates The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors have declared that no competing interests exist.Ethics StatementN/AData AvailabilityAll data are freely available without restriction.

Eph receptor tyrosine kinases and their membrane-bound ligand ephrins form an essential cell communication system. Both ephrin classes have been shown to localize within cell surface lipid rafts, yet regulate different biological processes. In order to provide insight into this distinct behavior, we examined ephrin-A5 and B1 localization and signaling in murine fibroblasts and tissues. Results indicated that ephrin-A5 was constitutively present in detergent-resistant membrane fractions, while ephrin-B1 displayed translocation to membrane fractions upon stimulation. Ephrin-A5 and B1 were present in detergent-resistant membrane fractions with different buoyancies in vitro and in different raft fractions in vivo. Moreover, ephrin-A5 and B1 differentially influenced actin reorganization. Finally, microarray analysis revealed unique patterns of gene expression between the two ephrin classes. We thus demonstrate that distinct localization and compartmentalization provide insight into the subcellular basis for differential signaling observed in ephrin-A and B classes.

The flavonoid quercetin 3-glucoside (Q3G) protected SH-SY5Y, HEK293, and MCF-7 cells against hydrogen peroxide-induced oxidative stress. cDNA microarray studies suggested that Q3G-pretreated cells subjected to oxidative stress up-regulate the expression of genes associated with lipid and cholesterol biosynthesis. Q3G pretreatment elevated both the expression and activation of sterol regulatory element-binding protein-2 (SREBP-2) only in SH-SY5Y cells subjected to oxidative stress. Inhibition of SREBP-2 expression by small interfering RNA or small molecule inhibitors of 2,3-oxidosqualene:lanosterol cyclase or HMG-CoA reductase blocked Q3G-mediated cytoprotection in SH-SY5Y cells. By contrast, Q3G did not protect either HEK293 or MCF-7 cells via this signaling pathway. Moreover, the addition of isopentenyl pyrophosphate rescued SH-SY5Y cells from the inhibitory effect of HMG-CoA reductase inhibition. Last, Q3G pretreatment enhanced the incorporation of [(14)C]acetate into [(14)C]cholesterol in SH-SY5Y cells under oxidative stress. Taken together, these studies suggest a novel mechanism for flavonoid-induced cytoprotection in SH-SY5Y cells involving SREBP-2-mediated sterol synthesis that decreases lipid peroxidation by maintaining membrane integrity in the presence of oxidative stress.

In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeoteSulfolobus solfataricusto UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.

The chemokine GRO-alpha (CXCL1) has been found to mediate the proliferation of glia progenitor cells during neural development. As malignant gliomas are thought to arise from glia progenitors or their differentiated counterparts, astrocytes or oligodendrocytes, we have investigated whether GRO-alpha regulates the tumor characteristics of glioma cells. We found first that resected glioma specimens were strongly immunoreactive for GRO-alpha expression in cells with the morphology of tumor cells. In culture, the U251 glioma line transfected to overexpress GRO-alpha had elevated levels of motility and invasiveness. GRO-alpha transfectants increased their expression of several proteins associated with migratory behavior, including matrix metalloproteinase-2, beta1-integrin and SPARC. The implantation of GRO-alpha glioma clones into the brain of nude mice caused the early demise of mice and this was associated with the formation of larger intracerebral tumors when compared with mice implanted with vector control lines. These results implicate GRO-alpha in gliomas and suggest that the dysregulation of a glia proliferative factor contributes to tumorigenesis. Targeting GRO-alpha may be a useful therapeutic tool to control brain tumor biology.

Granulosa cell tumors (GCTs) of the ovary are relatively rare and account for

Having the complete human genomic sequence poses a new challenge: to use genomic structural information to display and analyze biological processes on a genome-wide scale to assign gene function. DNA microarrays are a miniaturized, ordered arrangement of nucleic acid fragments from individual genes located at defined positions on a solid support, enabling the analysis of thousands of genes in parallel by specific hybridization. This review describes technical aspects, discusses relevant applications, and suggests factors affecting the use of this technology and how it fits in the grand scheme of meeting the needs of the postgenomic era.

Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.

Preimplantation embryos from a variety of mammalian species contrast markedly in their response to culture in vitro. Murine preimplantation embryos display a wider tolerance than other mammalian species to culture environments, and this has contributed to the development of several effective defined culture media. Embryo coculture on somatic cells remains the most effective method of supporting reasonable rates of bovine preimplantation development in vitro. The patterns of gene expression for several antioxidant enzymes during preimplantation murine and bovine development were examined by use of the reverse transcription-polymerase chain reaction technique to determine whether the differential developmental capacity of mammalian preimplantation embryos in culture may reflect variations in the patterns of expression for a series of antioxidant enzymes. Transcripts for catalase, CuZn-containing superoxide dismutase (CuZn-SOD), Mn-SOD, glutathione peroxidase (GPX), and glutamylcysteine synthetase (GCS) were detected in mouse embryos at all stages of development regardless of in vivo or in vitro development. Preimplantation cow embryos produced by in vitro procedures expressed mRNAs for catalase, CuZn-SOD and GPX, whereas transcripts for Mn-SOD were not detected at any stage. GCS transcripts, although present in stages up to the morula, were not detected in cow blastocysts. Analysis of antioxidant gene expression in both bovine primary oviductal cell monolayer cultures and nonattached, ciliated oviductal cell vesicle cultures revealed a constitutive pattern of expression of all five enzymes for the 8-day culture interval. These experiments suggest that differences in gene expression may contribute to the variation in the ability of embryos to develop in vitro with respect to levels of oxygen and dependence on coculture.

Several growth factor ligand and receptor gene products have been shown to play roles during preimplantation mammalian development. Genes for insulin-like growth factors (IGFs), transforming growth factors (TGFs), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and receptors for insulin, IGF, PDGF, TGF alpha and epidermal growth factor (EGF) are expressed by early embryos of several species including mouse, rat, cow and sheep. Roles of growth factors during early development have been demonstrated by addition of purified growth factors to culture medium or by molecular genetic techniques that interfere with gene expression. In this way, it has been shown that successful development of the blastocyst is dependent on the action of epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Recent experiments show that both LIF and EGF stimulate secretion of urokinase-type plasminogen activator (uPA) and gelatinase B/matrix metalloproteinase-9 (MMP-9) in day 7 mouse blastocyst outgrowths. At the same time, tissue inhibitors of MMPs (TIMPs) are also expressed by embryonic, decidual and uterine tissues during the implantation process. It appears that LIF may act directly or indirectly, by inducing the expression of other cytokines, to regulate the temporal and spatial production and activity of proteases and protease inhibitors to create a favourable environment for implantation.

The pattern of expression of major histocompatibility complex class I H-2K in early mouse embryos was determined through reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization methods. H-2K transcripts were detected in all stages of preimplantation embryos from oocyte to blastocyst. In the blastocyst, transcripts were found in both trophectoderm and inner cell mass. Quantitative RT-PCR revealed that in CD-1 mice H-2Kq transcript levels per oocyte/embryo were 71 +/- 13 in oocyte, 150 +/- 9 in 2-cell embryos, 220 +/- 22 in 8-cell embryos, and 560 +/- 86 in blastocysts. By RT-PCR, H-2Kq mRNA was also detected in postimplantation staged (7.5-, 8.5-, and 9.5-day) embryos. Examination of components of the 7.5-day conceptus revealed the highest levels in the ectoplacental cone (EPC), followed by the decidua, and finally the embryo. The spatial distribution of H-2K transcripts in decidua was very heterogeneous, whereas the low levels in the embryo were found to be uniform throughout. Both cell types of the EPC contained H-2K transcripts. In the 9.5-day embryo, high transcript levels were observed over the developing liver above a background of constitutive expression everywhere else.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

The temporal patterns of expression of genes encoding insulin-like growth factor (IGF) ligands and receptors during very early development have been investigated in several laboratories in several different mammalian species. Both reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical techniques have been used to identify the time of appearance of gene transcripts or end-products. In preimplantation mouse embryos, IGF-II ligand and receptor gene activity is detectable as early as at the two-cell stage, the time when transcription from the embryonic genome is activated, but receptors for insulin and IGF-I are not detectable until the compacted eight-cell stage. Transcripts for insulin or IGF-I are not detectable in preimplantation mouse embryos, although the ligands are present in the reproductive tract. The pattern of IGF gene expression is not, however, identical in all mammalian species. In cow embryos, for example, transcripts for IGF-I and IGF-II ligands and receptors and insulin receptors have been detected at all stages of preimplantation development from mature oocyte to blastocyst (Watson et al., 1992). Attempts to quantitate transcript abundance in these early embryos are in progress in our laboratory. In the preimplantation mouse embryo, transcripts for several different IGF-binding proteins (IGFBP-2, -3, -4, and -6) have been detected by RTPCR procedures. In addition, transcripts for IGFBPs have been identified in RNA derived from cumulus cells, the ovary, the oviduct, the uterus, and the decidua. These findings suggest that the interactions of IGF ligands and receptors in preimplantation development might, indeed, be modulated by IGFBPs. Approaches to function of IGFs in preimplantation embryos have involved analysis of the stimulatory effects on metabolism and cell division when IGFs are added exogenously to embryos in culture in simple defined medium (for example, see Harvey and Kaye, 1991) or observations in the reduction in rate of development following interference with IGF expression (see Rappolee et al., 1992). In general, members of the insulin and IGF gene family of polypeptides have been shown to lead to an enhahancement of development in vitro in both laboratory and domestic mammalian species. © 1993 Wiley-Liss, Inc.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.

The spindle-shaped Sulfolobus virus SSV1 was the first of a series of unusual and uniquely shaped viruses isolated from hyperthermophilic Archaea. Using whole-genome microarrays we show here that the circular 15.5 kb DNA genome of SSV1 exhibits a chronological regulation of its transcription upon UV irradiation, reminiscent to the life cycles of bacteriophages and eukaryotic viruses. The transcriptional cycle starts with a small UV-specific transcript and continues with early transcripts on both its flanks. The late transcripts appear after the onset of viral replication and are extended to their full lengths towards the end of the approximately 8.5 h cycle. While we detected only small differences in genome-wide analysis of the host Sulfolobus solfataricus comparing infected versus uninfected strains, we found a marked difference with respect to the strength and speed of the general UV response of the host. Models for the regulation of the virus cycle, and putative functions of genes in SSV1 are presented.

Emerging evidence suggests involvement of the ephrin/Eph receptor system in tumourigenesis. Research on this new role has centred on the contribution of Eph receptors. In contrast, we focused on the elucidation of the role of ephrins, specifically ephrin A5. Results indicated an increase in invasive potential of ephrin A5-expressing murine fibroblasts, which was abolished by addition of a Src family kinase inhibitor. Furthermore, anchorage-independent growth was increased in ephrin A5-expressing cells. Stimulation with EphA5-Fc receptor increased colony size, but not colony number in ephrin A5 transfectants. Moreover, we observed morphogenetic transformation of ephrin A5-expressing 3T3 cells into a branching network when plated onto Matrigel. This behaviour was specific to ephrin A5 transfectants, as 3T3 cells expressing ephrin B1 displayed a phenotype similar to control 3T3 cells. We conclude that ectopic expression of ephrin A5 in murine fibroblasts elevates oncogenic potential, including increased invasive behaviour, anchorage-independent growth, and morphological transformation.

Having the complete human genomic sequence poses a new challenge: to use genomic structural information to display and analyze biological processes on a genome-wide scale to assign gene function. DNA microarrays are a miniaturized, ordered arrangement of nucleic acid fragments from individual genes located at defined positions on a solid support, enabling the analysis of thousands of genes in parallel by specific hybridization. This review describes technical aspects, discusses relevant applications, and suggests factors affecting the use of this technology and how it fits in the grand scheme of meeting the needs of the postgenomic era.

Stem cells that can give rise to neurons, astroglia, and oligodendroglia have been found in the developing and adult central nervous system (CNS) of rodents. Yet, their existence within the human brain has not been documented, and the isolation and characterization of multipotent embryonic human neural stem cells have proven difficult to accomplish. We show that the developing human CNS embodies multipotent precursors that differ from their murine counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cell characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, and oligodendrocytes when growth factors are removed. Subcloning and population analysis show their extensive self-renewal capacity and functional stability, their ability to maintain a steady growth profile, their multipotency, and a constant potential for neuronal differentiation for more than 2 years. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Finally, we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesioned striatum of adult rats. Tumor formation is not observed, even in immunodeficient hosts. Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigenetic stimulation. These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the development of clinical neural transplantation.

Activin and its cognate receptors are expressed during embryogenesis in the rapidly dividing cells of the basal forebrain ventricular zone. This finding prompted us to study the role of activin in regulating neurotransmitter phenotype expression and other aspects of the ventricular zone-derived progenitor cell differentiation. Although virtually ineffective alone, activin co-operated with fibroblast growth factor 2 to induce a rapid tyrosine hydroxylase-immunoreactivity in cultured ventricular zone progenitors. Northern analysis indicated that the increase in tyrosine hydroxylase-immunoreactivity was associated with increased tyrosine hydroxylase gene expression. Activin and fibroblast growth factor 2 action was specific to tyrosine hydroxylase, as it did not induce the expression of choline acetyltransferase, nor enhance the expression of glutamate decarboxylase. Cultures treated with the DNA replication marker bromodeoxyuridine revealed that both proliferating ventricular zone progenitors and their post-mitotic progeny were induced to express tyrosine hydroxylase. In these cultures, activin acted to reduce fibroblast growth factor 2 stimulated mitotic activity. Furthermore, activin permitted neuronal differentiation and survival of the ventricular zone progenitors after three days in vitro. Together these data demonstrate a novel role of activin and fibroblast growth factor 2 in regulating the fate of the embryonic basal forebrain ventricular zone progenitors.

Several proteinases from different multigene families have been implicated in the uterine invasion required for estab-lishment of pregnancy in some mammals. In this study, the expression of matrix metalloproteinase gelatinase B (MMP-9), urokinase-type plasminogen activator (uPA) and their inhibitors was investigated during early mouse embryo development. Transcripts for tissue inhibitors of metalloproteinases (TIMP-1,-2,-3) and uPA receptor were detected throughout pre- and peri-implantation develop-ment whilst MMP-9 and uPA mRNAs were first detected in peri-implantation blastocysts associated with the invasive phase of implantation. Through use of in situ hybridization, it was shown that MMP-9 transcripts were strongly expressed in the network of trophoblast giant cells at the periphery of implanting 7.5 day embryos and TIMP-3 transcripts were strongly expressed in the decidua imme-diately adjacent to the implanting embryo. uPA transcripts were preferentially expressed in the ectoplacental cone and its derivatives. Because these proteinases are regulated by growth factors and cytokines in other tissues, the effect of leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) on their activity was investigated. Both LIF and EGF, like the proteinases, have been implicated in peri-implantation development. Blastocysts collected on day 4 of pregnancy were cultured 2 days in TCM 199 + 10% fetal bovine serum to allow outgrowth followed by 24 hour culture in defined media containing either LIF or EGF. Conditioned media were assayed for uPA activity by a chromogenic assay and MMP activity by gelatin zymog-raphy. Both LIF and EGF stimulated uPA and MMP-9 activity in blastocyst outgrowths after 3 days of culture (day 7). Proteinase activity was assayed again at the 5th to 6th day of culture (day 9 to 10). EGF was found to have no effect whereas LIF decreased production of both pro-teinases. These results demonstrate that proteinase activity in early embryos can be regulated by growth factors and cytokines during the implantation process and, in particu-lar, they demonstrate the possible involvement of LIF in establishment of the correct temporal programme of pro-teinase expression.

A number of growth factor ligand and receptor genes are expressed by early embryos of the mouse, rat, cow, and sheep. Several growth factors have stimulatory effects on early embryos when added exogenously to the medium for culture of preimplanta- tion mammalian embryos. Growth factor supplemen- tation of simple defined medium for culture of early bovine embryos leads to a higher percentage of embryos that can cleave and develop beyond the eight- cell stage compared to those in un-supplemented medium. Cultured preimplantation bovine embryos do not express as broad a range of antioxidant enzymes as do mouse embryos. Bovine primary oviduct co- culture cells constitutively express the same set of antioxidant enzymes as the mouse embryo and may contribute to protection of the embryo against damage from free oxygen radicals during in vitro culture. Preliminary experiments to analyze growth factor gene expression in cloned bovine embryos indicate that nuclear transplantation alters the normal pattern of expression during early embryonic development.