Annealing Control Primer System Research Articles

Identification of differentially expressed genes in a pistillody common wheat mutant using an annealing control primer system.

HTS-1 is a new kind of pistillody wheat. All or parts of its stamen are transformed into pistils or pistil-like structures, and it has more seed sets per floret than normal wheat under normal cultivation conditions. To investigate the expression divergence in this mutant, an annealing control primer system was used to identify differentially expressed genes (DEGs) in the young spikelets. As a result, three DEGs, including HDB2, HGF2, and HCG4, were detected, with variable expression in HTS-1 and the control. After further confirmation using real-time reverse transcription polymerase chain reaction analysis, these genes were overexpressed in HTS-1 wheat. NGF2 was identified in the double ridge to floret differentiation stages; HDB2 and HCG4 were identified in the stage of pistil and stamen-differentiating. Therefore, we inferred that the homeotic transformation of stamens into pistil-like structures occurred during the early stage of stamen development. Sequence alignment analysis revealed that HDB2 encodes a putative protein of 189 amino acids, with high homology to the DEAD-box ATP-dependent RNA helicase, and HCG4 was identical to the Chinese spring wheat cDNA clone predicted protein according to GenBank. However, NGF2 was not found to have significant similarity to any reported proteins, suggesting it is a new functional gene in wheat. The results suggest that HDB2, HCG4, and HGF2 are minor genes contributing to pistillody trait formation in HTS-1.

Identifying cotton (Gossypium hirsutum L.) genes induced in response to Aspergillus flavus infection

Development of aflatoxin-resistant cotton against Aspergillus flavus is handicapped by the lack of resistance source in available germplasm. Genetic engineering warrants for identification of resistance-associated genes in cotton. As a first step toward this, we isolated 44 differentially expressed genes (DEGs) in response to A. flavus infection, using annealing control primer system. Different functional classification of the DEGs suggested a complex and multi-factorial plant–fungus interaction. Eight DEGs, including transcription factors, kinase, and downstream stress responsive genes, showed a tissue- and time-dependent differences in their expression. The upregulated genes can be used as transgenes and/or functional markers for breeding aflatoxin-resistant cottonseed.

Identification of differentially expressed genes induced by energy restriction using annealing control primer system from the liver and adipose tissues of broilers

Female Arbor Acre broilers were divided into 2 groups at 18 d of age. One group of chickens had free access to feed (AL), and the other group of chickens had 30% energy restriction (ER). Adipose and hepatic RNA samples were collected at 48 d of age. We employed an accurate reverse-transcription (RT) PCR method that involves annealing control primers to identify the differentially expressed genes (DEG) between ER and AL groups. Using 20 annealing control primers, 43 differentially expressed bands (40 downregulated and 3 upregulated in the ER group) were detected from the hepatic tissue, whereas no differentially expressed bands were detected from the adipose tissue. It seems that energy restriction could induce more DEG in hepatic tissue than that in adipose tissue and could result in more gene-expression downregulation in hepatic tissue. Eight DEG (6 known and 2 unknown genes) were gained from hepatic tissue and confirmed by RT-PCR, which were all supported by released expressed sequence tag sequences. Their expressions were all downregulated by energy restriction in hepatic tissues. Six known genes are RPL7, RPLP1, FBXL12, ND1, ANTXR2, and SLC22A18, respectively, which seem to play essential roles in the protein translation, energy metabolism, and tumor inhibition. The alterations of gene expression in 3 selected genes, including ND1 (P < 0.01), FBXL12 (P < 0.01), and RPLP1 (P < 0.05), were supported by real-time quantitative RT-PCR reaction. Our data provide new insights on the metabolic state of broilers changed by energy restriction.

Identification of differentially expressed genes in three-pistil mutation in wheat using annealing control primer system

The common wheat line three-pistil (TP) is a valuable mutant for wheat breeding. The TP mutation has normal spike morphology; however, it only produces three pistils per floret. Therefore, it has potential to increase the grain number per spike. In order to determine the underlying molecular mechanism, an annealing control primer system was used to identify the different expressed genes in three-pistil mutation. Using 120 arbitrary ACP primers, we identified three differentially expressed genes in young spikes between two near-isogenic lines (i.e., Chuanmai 28 TP and Chinese Spring TP) and their recurrent parents. We tentatively designated the three differentially expressed genes as DETP-1, DETP-2, and DETP-3. DETP-1 showed similar function with maize cytoplasmic membrane protein, which is involved in cell division in bacteria. DETP-3 is homologous to maize endo-1, 4-beta-glucanase (EGases), which is associated with plant development, cell wall loosening, stem flowering, and root expansion. DETP-2 showed no significant hit with any sequence found in the database and translates unknown protein. These genes would likely play an important role in determining the three pistils trait in wheat.

Identification of CCL1 as a Gene Differentially Expressed in CD4+T Cells Expressing TIM-3

BackgroundT cell immunoglobulin mucin containing molecule (TIM)-3 is expressed in differentiated Th1 cells and is involved in the suppression of the cytokine production by these cells. However, the regulation of the expression of other T cell genes by TIM-3 is unclear. Herein, we attempted to identify differentially expressed genes in cells abundantly expressing TIM-3 compared to cells with low expression of TIM-3.MethodsTIM-3 overexpressing cell clones were established by transfection of Jurkat T cells with TIM-3 expression vector. For screening of differentially expressed genes, gene fishing technology based on reverse transcription-polymerase chain reaction (RT-PCR) using an annealing control primer system was used. The selected candidate genes were validated by semi quantitative and real-time RT-PCR.ResultsThe transcription of TIMP-1, IFITM1, PAR3 and CCL1 was different between TIM-3 overexpressing cells and control cells. However, only CCL1 transcription was significantly different in cells transiently transfected with TIM3 expression vector compared with control cells. CCL1 transcription was increased in primary human CD4+ T cells abundantly expressing TIM-3 but not in cells with low expression of TIM-3.ConclusionCCL1 was identified as a differentially transcribed gene in TIM-3-expressing CD4+ T cells.

Identification of differentially expressed genes using an annealing control primer system in stage III serous ovarian carcinoma

BackgroundMost patients with ovarian cancer are diagnosed with advanced stage disease (i.e., stage III-IV), which is associated with a poor prognosis. Differentially expressed genes (DEGs) in stage III serous ovarian carcinoma compared to normal tissue were screened by a new differential display method, the annealing control primer (ACP) system. The potential targets for markers that could be used for diagnosis and prognosis, for stage III serous ovarian cancer, were found by cluster and survival analysis.MethodsThe ACP-based reverse transcriptase polymerase chain reaction (RT PCR) technique was used to identify DEGs in patients with stage III serous ovarian carcinoma. The DEGs identified by the ACP system were confirmed by quantitative real-time PCR. Cluster analysis was performed on the basis of the expression profile produced by quantitative real-time PCR and survival analysis was carried out by the Kaplan-Meier method and Cox proportional hazards multivariate model; the results of gene expression were compared between chemo-resistant and chemo-sensitive groups.ResultsA total of 114 DEGs were identified by the ACP-based RT PCR technique among patients with stage III serous ovarian carcinoma. The DEGs associated with an apoptosis inhibitory process tended to be up-regulated clones while the DEGs associated with immune response tended to be down-regulated clones. Cluster analysis of the gene expression profile obtained by quantitative real-time PCR revealed two contrasting groups of DEGs. That is, a group of genes including: SSBP1, IFI6 DDT, IFI27, C11orf92, NFKBIA, TNXB, NEAT1 and TFG were up-regulated while another group of genes consisting of: LAMB2, XRCC6, MEF2C, RBM5, FOXP1, NUDCP2, LGALS3, TMEM185A, and C1S were down-regulated in most patients. Survival analysis revealed that the up-regulated genes such as DDAH2, RNase K and TCEAL2 might be associated with a poor prognosis. Furthermore, the prognosis of patients with chemo-resistance was predicted to be very poor when genes such as RNase K, FOXP1, LAMB2 and MRVI1 were up-regulated.ConclusionThe DEGs in patients with stage III serous ovarian cancer were successfully and reliably identified by the ACP-based RT PCR technique. The DEGs identified in this study might help predict the prognosis of patients with stage III serous ovarian cancer as well as suggest targets for the development of new treatment regimens.

Molecular Cloning of Proliferating Cell Nuclear Antigen and Its Differential Expression Analysis in the Developing Ovary and Testis of Penaeid ShrimpMarsupenaeus japonicus

To understand the molecular mechanisms of gonadal development and maturation in penaeid shrimp Marsupenaeus japonicus, eight differentially expressed genes were obtained using a modified annealing control primer system. One of these genes is a proliferating cell nuclear antigen (PCNA). Bioinformatics analyses showed that full-length cDNA of M. japonicus PCNA (mjPCNA) consists of 75 bp of 5' untranslated region, 783 bp of coding region, and 65 bp of 3' untranslated region (excluding the polyA tail), encoding a protein of 260 amino acids with a predicted molecular mass of 28.85 kDa and an isoelectric point of 4.59. Real-time polymerase chain reaction analyses demonstrated that the gene expression level changed significantly in the developing testis and ovary. In stage 1 of ovary and testis, mjPCNA showed its lowest level during development and reached its highest expression level in stage 2 of ovary and testis. In stages 4 and 5 of ovary and the stage 3 of testis, mjPCNA held a steady expression level. Data suggest that PCNA plays an important role in the testis and ovary development, especially in the process of mitosis and meiosis.

Identification of differentially expressed genes in the developmental stages from olive flounder Paralichthys olivaceus using an annealing control primer system

We employed a new and improved differential display reverse transcription–polymerase chain reaction (DDRT-PCR) method, which involves annealing control primers (ACPs), to identify the genes that are specifically or prominently expressed in olive flounder (Paralichthys olivaceus) juveniles (35 days post-hatch; dph) compared to larval-stage (dph 21) flounder. Using 60 ACPs, we identified eight differentially expressed genes (DEGs) and basic local alignment search tool (BLAST) searches revealed eight known genes. Gene expression levels were confirmed by RT-PCR. Phosphoglucose isomerase (PGI) was highly expressed at 21 dph, while nephrosin, myosin light chain (MLC), myosin heavy chain (MHC), carboxypeptidase A, chymotrypsin B, fish-egg protein, and matrix protein were expressed at 35 dph. PGI, MLC, and MHC expression was further analyzed by RT-PCR. The differentially expressed genes identified in this study may provide insights into the molecular basis of development in olive flounder.

Identification of immune responsible fibrinogen beta chain in the liver of large yellow croaker using a modified annealing control primer system

In this article, we used a modified ACP system (mACP) developed in our laboratory to analyze differentially expressed genes in the liver of large yellow croaker, Pseudosciaena crocea (Richardson). By using 20 pairs of mACPs, 7 differentially expressed genes were obtained. One of the genes we identified encodes for a fibrinogen beta chain ( FGB). The full-length cDNA of FGB was 1645 bp, including 5 bp of 5′ untranslated region (5′-UTR), 1479 bp of open reading frame (ORF), and 161 bp of 3′-UTR. The ORF was capable of encoding 492 amino acids with an estimated molecular mass of 55.6 kDa, giving it a predicted pI of 5.94. The deduced amino acid sequence included an FGB profile (V 238-Y 488) and an FGB family signature (WWYNRCHSANPNG). Multiple sequence alignments indicated that the large yellow croaker FGB showed homology with FGB sequences of other species (45–77% identity). Real time PCR analysis demonstrated that the expression of FGB in the liver of large yellow croaker injected with Vibrio parahaemolyticus was significantly ( P < 0.05) lower than that of the control group at 8 d, which confirmed the expression patterns of the results of mACP differential display.

Mac-2 binding protein and galectin-3 expression in mucinous tumours of the ovary: an annealing control primer system and immunohistochemical study

We used a new differential display method, the annealing control primer (ACP) system, to analyse the differentially expressed genes in mucinous ovarian tumours. To verify the corresponding target gene, immunohistochemical staining was performed on various epithelial tumours of the ovary. The ACP-based reverse transcriptase-polymerase chain reaction revealed that 21 genes were upregulated in the mucinous ovarian adenoma and 14 genes were upregulated in the mucinous ovarian carcinoma. Among them, we selected one upregulated gene, the Mac-2 binding protein (Mac-2 BP), and verified the expression of the Mac-2 BP and its ligand, galectin-3, in a variety of epithelial ovarian tumours by immunohistochemistry. Positive expression of the Mac-2 BP was significantly higher in the mucinous ovarian tumours compared to the other epithelial tumours. Mac-2 BP expression was significantly increased in the borderline and malignant tumours compared to the benign tumours. Galectin-3 expression was more frequent in clear cell carcinomas, serous tumours and mucinous tumours than in endometrioid and transitional tumours. However, there were no differences in galectin-3 expression in comparisons among benign, borderline and malignant mucinous and serous tumours. These data indicate that the Mac-2 BP may play a role in the development and progression of mucinous ovarian tumours.

Identification of Differential Gene Expression in Juvenile vs. Mature Leaves of Pear (Pyrus pyrifolia) by Using Annealing Control Primer

The identification of genes specifically expressed at a developmental stage would provide insights into many characteristic features of each of the developmental stages and introduction of molecular markers linked to important agronomic traits. However, the current methods employed to identify genes with specific expression are labor-intensive and suffer from high rates of false positives. Here, we employed a new and accurate reverse transcription-polymerase chain reaction method using annealing control primers (ACP) to identify genes that are preferentially expressed in juvenile or mature leaves of pears (Pyrus pyrifolia L.). We identified and sequenced four of these differentially expressed genes (DEGs). Basic Local Alignment Search Tool searches revealed that one was a hypothetical gene and three genes encoded protein with high similarity to the known genes. These DEGs were further characterized using real-time quantitative PCR to assess their expression pattern. This report suggests that the ACP system is a rapid and accurate method for the identification of development-specific genes in woody plants such as pears, of which a few genes are available.

Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine

Pine wilt disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, has destroyed huge areas of pine forest in East Asia, including Japan, China and Korea. No protection against PWN has been developed, and the responses of pine trees at the molecular level are unrecorded. We isolated and analyzed upregulated or newly induced genes from PWN-inoculated Japanese red pine (Pinus densiflora Sieb. et Zucc.) by using an annealing control primer system and suppression subtractive hybridization. Significant changes occurred in the transcript abundance of genes with functions related to defense, secondary metabolism and transcription, as the disease progressed. Other gene transcripts encoding pathogenesis-related proteins, pinosylvin synthases and metallothioneins were also more abundant in PWN-inoculated trees than in non-inoculated trees. Our report provides fundamental information on the molecular mechanisms controlling the biochemical and physiological responses of Japanese red pine trees to PWN invasion.

Identification of differential gene expression for Kr1 gene in bread wheat using annealing control primer system

Intergeneric hybridization is an important strategy to introgress alien genes into common wheat for its improvement. But presence of cross ability barrier mechanism regulated by Kr1 gene played a major destructive role for hybridization than other reported genes. In order to know the underlying molecular mechanism and to dissect out this barrier, a new annealing system, ACP (anneling control primer) system was used in chromosome 5B (containing Kr1 gene) specific Recombinant Inbred Line (RIL) population. Two differentially expressed fragments for Kr1 gene was identified, cloned and sequenced. Further the expression was confirmed by northern blotting analysis. Sequence analysis of the resulted clones revealed classes of putative genes, including stress responsive and signal transduction.

Identification of replicative senescence-associated genes in human umbilical vein endothelial cells by an annealing control primer system

Cellular senescence is regulated by specific genes in many organisms. The identification and functional analysis of senescence-associated genes could provide valuable insights into the senescence process. Here, we employed a new and improved differential display reverse transcription-polymerase chain reaction (DDRT-PCR) method that involves annealing control primers (ACPs) to identify genes that are differentially expressed in human umbilical endothelial cells during replicative senescence. Using 120 ACPs, we identified 31 differentially expressed genes (DEGs). Basic local alignment search tool (BLAST) search revealed 29 known genes and two unknown genes. Expression levels of the 29 known genes were confirmed by real-time quantitative RT-RCR and by Western blotting for eight of these genes. CD9 antigen, MHC class I chain-related sequence A (MICA) and cell division cycle 37 homolog (CDC37) were up-regulated, and bone morphogenetic protein 4 (BMP4), dickkopf-1 (DKK1), and transcription factor 7-like 1 (TCF7L1) were down-regulated in old cells. Treatment with recombinant human MICA caused a decrease in cell proliferation and an increase in senescence-associated β-galactosidase staining. Further analysis of differentially expressed genes may provide insights into the molecular basis of replicative senescence and vascular diseases associated with cellular senescence.

Tenascin-X and leukemia inhibitory factor receptor are down-regulated in leiomyoma compared with normal myometrium.

Uterine leiomyomas are the most common tumor of the uterus. But the molecular causes of uterine leiomyoma remain unclear. We conducted the current investigation in order to elucidate the molecular mechanisms in the development of uterine leiomyoma. We employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method that involved annealing control primers (ACPs) to identify the genes that are differently expressed in uterine leiomyoma. Using 120 ACPs, we identified and sequenced 14 differently expressed genes (DEGs) in uterine leiomyoma compared with normal myometrium. Basic Local Alignment Search Tool (BLAST) searches were performed to examine the known functions of these genes associated with uterine leiomyoma. We confirmed differently expressed patterns in more cases using the RT-PCR method. We also detected two novel genes, Tenascin-X and Leukemia Inhibitory Factor Receptor (LIFR), which had not yet been reported to have any functions associated with uterine leiomyoma. RT-PCR confirmation shows that both of these two genes are down-regulated in uterine leiomyoma. Our results suggest that Tenascin-X and LIFR may play a role in the development of uterine leiomyoma. Although further studies are required to establish the precise mechanisms with which these genes are involved in the genesis of uterine leiomyoma, the present research is significant in that it is the first study which detects down-regulated novel genes in uterine leiomyoma using the ACP system.

Screening Genes Expressed by Rhizobium vitis Inoculation and Salicylic Acid Treatment in Grapevines Using GeneFishing

To search for specifically expressed genes during resistant responses against Rhizobium vitis infection from grapevine leaves, the project employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method using annealing control primers (ACPs) to minimize the incidence of false-positive clones. Twenty ACPs were used to identify and sequence 31 differentially expressed genes (DEGs). Basic Local Alignment Search Tool (BLAST) searches revealed that 27 of these genes are known. Of the known genes, six were selected and further characterized using semi-quantitative RT-PCR analysis to assess their specific expression in plants infected with R. vitis. Results of the analysis suggest that the ACP system is a very useful tool for identifying disease resistance-specific genes in grapevines. Further analysis of the cloned differentially expressed genes will provide insights into the molecular basis of disease resistance expression.

Identification and Isolation of Differentially Expressed Gene in Response to Cold Stress in a Green Alga, Spirogyra varians (Zygnematales)

In plants, cold adaptation is characterized by various morphological and physiological transitions. Plant survives cold stress using various methods. Some plants produce antifreeze protein (Guy et al. 1985) or change the composition of lipid and carbohydrate in the cell (Guy 1990) to increase the ability to endure cold stress; but most plants regulate expression of certain gene group to adjust to cold stress (Thomashow 1990). There are many reports on the cold stress regulated genes of higher plants as wheat, barley and Arabidopsis spp. (Graham and Patierson 1982; Cattivelli and Bartels 1990; Hajela et al. 1990; Gilmour et al. 1992; Danyluk et al. 1994; Houde et al. 1995; Pearce et al. 1998), but little is known about the cold stress genes and cold acclimation mechanism in algae. Cold shock can damage a freshwater alga much more seriously than higher plants because most of over-wintering freshwater algal cells are directly exposed to freezing waters during winter time. In order to maintain their functions under cold stress, freshwater alga must have been developing some unique cold acclimation mechanisms. Spirogyra varians grows in shallow ponds in Korea from late January to April. The water repeats freezing and thawing during this period. This species is often found growing under the ice. When blue light is given, plants move toward to the light source and this phototrophic movement is affected by temperature (Kim et al. 2005). Differential displayed (DD)-PCR has been developed to identify and isolate differentially expressed genes (Liang and Pardee 1992) and is extensively applied to various ranges of gene expression analyses because of its effectiveness and convenience. One of the advantages of this technique is that it requires only small amount of RNA since this technique is PCR-based. However, relatively high chance to have false-positive acts is a major handicap in this technique. Many efforts have been attempted to improve the specificity of DD-PCR (Liang et al. 1993, 1994). Recently modification to eliminate falsepositive acts has been developed by increasing the annealing specificity with specially designed annealing control primers (ACP) system (Hwang et al. 2003; Kim et al. 2004). In this study, we analyzed the cold stress associated genes in Spirogyra varians by differential expression gene (DEG) technique using ACP system. A coldAlgae Volume 22(2): 131-139, 2007

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES AFTER HEAT SHOCK IN ISOLATED RAT AORTA

1. In a previous study, we demonstrated that heat shock augments vascular contractility through the stress response. 2. The current study was designed to identify differentially expressed genes after heat shock by using a novel annealing control primer (ACP) system, which was developed recently to identify authentic genes. 3. Rat aortic rings were mounted in organ baths, exposed to 42 degrees C for 45 min and harvested 4 h after the end of heat shock. Total RNA were used for amplification by the reverse transcriptase-polymerase chain reaction (RT-PCR) with ACP system. Differentially amplified PCR products were sequenced, searched against the GenBank and confirmed by RT-PCR. 4. Genes for connective tissue growth factor, stress-inducible protein 1 and heat shock protein 25 were upregulated, whereas a gene for interferon regulatory factor 1 was downregulated. Immunohistochemistry revealed upregulation of the phosphorylated form of Hsp25 in aortic rings after heat shock. 5. These results suggest that phosphorylated Hsp25 plays a pivotal role in the augmentation of vascular contraction after heat shock.

Identification of differentially expressed genes in the testis of Sprague-Dawley rats treated with di( n-butyl) phthalate

The aim of this study was to identify the di( n-butyl) phthalate (DBP)-induced differentially expressed genes (DEGs) using a novel annealing control primer system in the testes of Sprague-Dawley male rats. Animals (4 weeks of age) were administered orally either corn oil only (vehicle control) or DBP (250, 500, or 750 mg/kg/day) for 30 days. Total RNA was isolated from the rat testes and GeneFishing PCR was used to determine the differential gene expression levels. Using this technique, a total of 59 DEG mRNA fragments were observed in the testes treated with DBP 750 mg/kg/day compared to vehicle control. Of these 59 genes, 31 genes were significantly altered after exposing rats to high dose DBP (750 mg/kg/day), and their sequences cloned. Based on the Basic Local Alignment Search Tool (BLAST), 4 expressed sequence tags (EST), 27 cloned genes ( Insl3, pgrp, H1SHR, etc.) and 3 genes (LDHA, lactate dehydrogenase A; Spag4, sperm associated antigen 4 and PBR, peripheral-type benzodiazepine receptor) were found to be involved in spermatogenesis and steroidogenesis. In addition, the expression patterns of the steroidogenesis-related genes such as scavenger receptor class B-1 (SR-B1), steroidogenic acute regulated protein (StAR), P450 side chain cleavage (P450scc), CYP17, and CYP19 were further analyzed by RT-PCR. Significant increases in the mRNA levels of steroidogenesis-related genes (PBR, SR-B1, StAR, P450scc, and CYP17) were observed in the high dose DBP-treated rats. However, DBP significantly decreased the CYP19 mRNA levels compared with controls. DBP (750 mg/kg/day) significantly increased the TR-α1 and PPAR γ expression in testes, whereas the AR and ERβ protein levels were significantly reduced in the same group. These data indicate that the steroidogenesis- or spermatogenesis-related genes identified in this study may provide insights into the molecular mechanisms underlying environmental pollutants-mediated male infertility.

Annealing control primer system identifies differentially expressed genes in blastocyst-stage porcine parthenotes

There is very little information available on stage-specific gene expression during early embryo development, particularly in the pig. Here, we accurately identified the genes that are specifically or prominently expressed in parthenogenetic porcine blastocysts as compared with 2-cell stage embryos. We accomplished this by using a PCR technology regulated by annealing control primers (ACPs). By utilizing 120 ACPs, a total of 46 expressed sequence tags (ESTs) of genes that are differentially expressed in blastocysts as compared with 2-cell stage embryos were cloned and sequenced. The cloned genes or ESTs all exhibited significant sequence similarity with known genes or ESTs of other species. Of the known genes, six genes [renin-binding protein (RNBP), BMDP, solute carrier family 25 (SLC25A6), MTHFD1, TRK-fused gene (TFG), spermidine synthase (SRM)] were selected and their stage-specific expression levels in porcine parthenotes were determined by real-time quantitative polymerase chain reaction at the 1-, 2-, 4-cell, morula and blastocyst stages. While RNBP, BMDP, SLC25A6, MTGFD1 and SRM were highly expressed only at the blastocyst stage, TFG was highly expressed at the 1-cell stage, then declined after genomic activation, high levels of expression being again detected at the morula and blastocyst stages. This analysis suggests that the ACP system is an effective tool for use in the identification of stage-specific genes in small numbers of porcine parthenotes. Examination of the genes differentially expressed in the blastocyst, which we have identified here, will provide insight into the molecular basis of preimplantation development.