Control Primer Research Articles

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 potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.

Use of the Non-electrophoretic Method to Detect Testis Specific Protein Gene for Sexing in Preimplantation Bovine Embryos

Testis-specific protein (TSPY) is a Y-specific gene, with up to 200 copy numbers in bulls. In order to make bovine embryo sexing under farm condition more feasible, the possibility of using a non-electrophoretic method to detect the TSPY gene for sexing bovine early embryos was examined. Primers were designed to amplify a portion of the TSPY gene and a common gene as an internal control primer. PCR optimization was carried out using a DNA template from bovine whole blood. Furthermore, embryo samples were diagnosed by this method and the sexing results were contrasted with those of the Loop-Mediated Isothermal Amplification (LAMP) method. The results showed that TSPY was as reliable a sexing method as LAMP. Forty-three morula and blastocyst embryos collected from superovulated donor dairy cattle were sexed by this method, and twenty-one embryos judged to be female embryos were transferred non-surgically to recipients 6 to 8 days after natural estrus. Out of 21 recipients, 9 were pregnant (42.86%) and all delivered female calves. The results showed that the sex predicted by this protocol was 100% accurate. In conclusion, the TSPY gene was a good male specific marker and indicated that a non-electrophoretic method was feasible and accurate to detect the TSPY gene for sexing preimplantation bovine embryos.

A Functional Enhancer of Keratin14 Is a Direct Transcriptional Target of ΔNp63

Keratin14 (K14) is a prototypic marker of dividing basal keratinocytes where its gene is transcribed at high levels. Transcriptional regulation of K14 is governed by an evolutionarily conserved functional enhancer marked by DNase 1 hypersensitive sites present upstream of the gene. This enhancer is sufficient to confer epidermal-specific gene expression, which is mediated in part by binding of members of activator protein-2 (AP)-2, AP-1, Ets, and Sp1 families of transcription factors. Here we provide evidence that a keratinocyte-specific nuclear protein identified as deltaNp63 binds to a conserved motif within this enhancer. Interestingly, the selective expression profile of deltaNp63 in various cell lines correlates with both the nuclear complex and the expression of K14. Biochemical studies reveal that deltaNp63 can bind to a specific DNA sequence present in the K14 enhancer and this binding leads to transactivation. In addition, chromatin immunoprecipitation experiments with deltaNp63-specific antibodies demonstrate that the enhancer is occupied by deltaNp63 in cultured keratinocytes and in mouse skin epidermal cells in vivo. Finally, we show that ectopic expression of either p63 isoform (deltaN or TA) can induce de novo expression of K14. These studies provide a potential mechanism by which deltaNp63 directly governs the expression of K14 in a keratinocyte-specific manner.

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 Using Annealing Control Primer-based GeneFishing in Human Squamous Cell Cervical Carcinoma

Aims To compare different gene expression patterns between squamous cell cervical carcinoma (SCC) and normal cervical tissue in Korean women and to identify those genes that are specifically or predominantly expressed in SCC by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR). Materials and methods Cervical cancer specimens were obtained from patients enrolled at the Department of Obstetrics and Gynecology, Kang Nam St. Mary's Hospital, Catholic University of Korea. We used a common reference that was mixed with an equal amount of RNA extracted from patients without cervical cancer. The profiles of expressed genes were compared between the SCC and normal cervix identified using GeneFishing differentially expressed gene kits, screened by a BLAST search, and confirmed by semi-quantitative reverse transcription-PCR (RT-PCR). Results Almost 100 differentially expressed genes were identified in the control and SCC samples. Using 60 arbitrary ACPs, 50 differentially expressed genes were identified, and 30 up-regulated and 20 down-regulated expressed genes were sequenced. Among 50 clones selected by ACP-based GeneFishing PCR, six genes with different expression patterns were determined and confirmed by semi-quantitative RT-PCR. The functional roles of two up-regulated genes, fibrillarin and calgranulin A, and one down-regulated gene, clusterin, were previously identified. However, the functional roles of two up-regulated genes and one down-regulated gene were not identified. Conclusion We identified distinctive gene expression profiles in Korean women with SCC using ACP-based GeneFishing PCR.

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.

Analysis of Human V-erbA Related EAR-3 Gene Expression between Transitional Cell Carcinoma and Normal Tissue in Bladder Cancer

Purpose: The prognosis of bladder cancer is related to tumor grade and stage. Because these pathological changes are preceded by molecular alterations, new molecular markers are needed in early diagnosis. New target molecular biomarkers can be differentially expressed genes (DEGs) between normal and cancer tissues. We tried to find a new DEG and demonstrated that it may be related to the development of the bladder cancer. Materials and Methods: Cancer tissues were obtained from 39 patients with urothelial cell carcinoma, treated by transurethral resection of tumor (TURB) since 2002. Normal bladder tissues were obtained from the same patients during TURB. We compared the mRNA profiles between normal and cancer tissues using annealing control primer (ACP)-based Genefishing PCR to identify the DEGs in normal and cancer tissues of one same patient. To validate the result of ACP-based GeneFishing PCR, reverse transcription-polymerase chain reaction (RT-PCR) was performed on those of 39 patients. Results: According to the result of ACP-based Genefishing PCR, EAR-3 gene was only present or markedly upregulated in normal tissue, compared with cancer tissues. The expression pattern that EAR-3 gene was downregulated in cancer tissues, irrespective of the clinicopathologic parameters was confirmed by RT-PCR in 39 patients. Conclusions: EAR-3 gene was downregulated in cancer tissues, irrespective of clinicopathologic parameters, compared with normal tissues in the bladder of the same patient. Therefore, we suggested that EAR-3 gene may be also play a role in bladder cancer development. (Korean J Urol 2007;48:915-920)

A randomized clinical trial comparing ‘one-step’ and ‘two-step’ orthodontic bonding systems

The primary objective of this prospective clinical trial was to assess the clinical bond failure rates of orthodontic brackets bonded using a self-etching primer (SEP), compared with brackets bonded using a conventional acid-etched technique with control adhesive (Transbond). A secondary aim was to investigate whether characteristics of the operator, patient or tooth bonded had any influence on bracket failure. Single-centre randomized controlled clinical trial. Thirty-four patients were bonded, each being randomly assigned to either the test or control adhesive. NHS Hospital Orthodontic Department, Chester, UK. Orthodontic patients requiring fixed appliance treatment. Bond failure. Failure rates over the initial 6-month period were 2.0% (Transbond) and 1.7% (SEP) with no statistically significant difference between the two groups. Over the duration of the fixed appliance treatment, bond failure rates increased, but remained acceptable at 7.4 % (TB) and 7.0% (SEP), respectively. When operator, patient and tooth characteristics were analysed, only the bracket location was found to be significant. Maxillary brackets were more likely to fail than mandibular brackets (RR 0.47%; 95% CI 0.22, 1.03). The failure rate for brackets in our study was low when compared with previous studies. Both the acid-etched control and self-etching primer in combination with adhesive pre-coated brackets were successful for clinical bonding. Their combined failure rate was lower than that reported in similar trials.

Nephrocan, a Novel Member of the Small Leucine-rich Repeat Protein Family, Is an Inhibitor of Transforming Growth Factor-β Signaling

In a search of new, small leucine-rich repeat proteoglycan/protein (SLRP) family members, a novel gene, nephrocan (NPN), has been identified. The gene consists of three exons, and based on the deduced amino acid sequence, NPN has 17 leucine-rich repeat motifs and unique cysteine-rich clusters both in the N and C termini, indicating that this gene belongs to a new class of SLRP family. NPN mRNA was predominantly expressed in kidney in adult mice, and during mouse embryogenesis, the expression was markedly increased in 11-day-old embryos at a time when early kidney development takes place. In the adult mouse kidney, NPN protein was located in distal tubules and collecting ducts. When NPN was overexpressed in cell culture, the protein was detected in the cultured medium, and upon treatment with N-glycosidase F, the molecular mass was lowered by approximately 14 kDa, indicating that NPN is a secreted N-glycosylated protein. Furthermore, transforming growth factor-beta (TGF-beta)-responsive 3TP promoter luciferase activity was down-regulated, and TGF-beta-induced Smad3 phosphorylation was also inhibited by NPN, suggesting that NPN suppresses TGF-beta/Smad signaling. Taken together, NPN is a novel member of the SLRP family that may play important roles in kidney development and pathophysiology by functioning as an endogenous inhibitor of TGF-beta signaling.

Interplay between Chromatin and Trans-acting Factors Regulating the Hoxd4 Promoter during Neural Differentiation

Correct patterning of the antero-posterior axis of the embryonic trunk is dependent on spatiotemporally restricted Hox gene expression. In this study, we identified components of the Hoxd4 P1 promoter directing expression in neurally differentiating retinoic acid-treated P19 cells. We mapped three nucleosomes that are subsequently remodeled into an open chromatin state upon retinoic acid-induced Hoxd4 transcription. These nucleosomes spanned the Hoxd4 transcriptional start site in addition to a GC-rich positive regulatory element located 3' to the initiation site. We further identified two major cis-acting regulatory elements. An autoregulatory element was shown to recruit HOXD4 and its cofactor PBX1 and to positively regulate Hoxd4 expression in differentiating P19 cells. Conversely, the Polycomb group (PcG) protein Ying-Yang 1 (YY1) binds to an internucleosomal linker and represses Hoxd4 transcription before and during transcriptional activation. Sequential chromatin immunoprecipitation studies revealed that the PcG protein MEL18 was co-recruited with YY1 only in undifferentiated P19 cells, suggesting a role for MEL18 in silencing Hoxd4 transcription in undifferentiated P19 cells. This study links for the first time local chromatin remodeling events that take place during transcriptional activation with the dynamics of transcription factor association and DNA accessibility at a Hox regulatory region.

Evaluation of three internal control primers for routine RTPCR plant virus detection

Evaluation of three internal control primers for routine RTPCR plant virus detection

Detection of the JAK2 V617F Mutation by LightCycler PCR and Probe Dissociation Analysis

A point mutation in the JAK2 gene, a member of the tyrosine kinase family, was recently identified and shown to be associated with several myeloproliferative disorders. Several studies identified the same JAK2 point mutation (1,849G>T), resulting in the substitution of a valine to phenylalanine at codon 617 (V617F). We developed a simple and sensitive method to detect this mutation via polymerase chain reaction and probe dissociation analysis using the LightCycler platform, and we compared this method to existing restriction fragment-length polymorphism, direct sequencing, and amplification refractory mutation system methods. We found that the LightCycler method offered advantages of speed, reliability, and more straightforward interpretation over the restriction fragment-length polymorphism and sequencing approaches.

Multiplex RT-PCR Assay for the Detection of Apple stem grooving virus and Apple chlorotic leaf spot virus in Infected Korean Apple Cultivars

To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the N-terminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RT-PCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor . This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.

Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast

We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.

ADP-Ribosylation Factor-Like 3 Is Involved in Kidney and Photoreceptor Development

ADP-ribosylation factor-like 3 (Arl3) is a member of a small subfamily of G-proteins involved in membrane-associated vesicular and intracellular trafficking processes. Genetic studies in Leishmania have shown that the Arl3 homolog is essential for flagellum biogenesis. Mutations in a related human family member, Arl6, result in Bardet-Biedl syndrome in humans, which is characterized by genital, renal, and retinal abnormalities, obesity, and learning deficits. As part of our large-scale phenotypic screen, mice deficient for the Arl3 gene were generated and analyzed. Arl3 (-/-) mice were born at a sub-Mendelian ratio, were small and sickly, and had markedly swollen abdomens. These mutants failed to thrive, and all died by 3 weeks of age. The (-/-) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, which is characteristic of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease. Absence of Arl3 was associated with abnormal epithelial cell proliferation and cyst formation. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration as early as postnatal day 14. These results are the first to implicate Arl3 in a ciliary disease affecting the kidney, biliary tract, pancreas, and retina.

Identification of differentially expressed genes involved in spine formation on seeds ofDaucus carota L. (carrot), using annealing control primer (ACP) system

Carrot seeds normally have surface spines. The availability of a spineless mutant would be agronomically beneficial, eliminating the current efforts to remove those spines pre-sowing. Furthermore, the identification of spine-specific genes would provide insights into spine development in wild-type carrot seed. This effort could be facilitated through the use of an annealing control primer (ACP) system. Here, we employed a new and accurate reverse transcriptionpolymerase chain reaction (RT-PCR) that involves ACPs for identifying genes of interest. With these techniques, 11 expressed sequence tags (ESTs) were obtained for cloning and sequencing the genes that are differentially expressed in wild-type spiny seeds, but not in the spineless mutant. In all, 7 cDNAs exhibited significant sequence similarity with known genes from other species. These included cell wall-associated hydrolase, tail fiber assembly protein, transcriptional regulatory protein, berberine bridge enzyme, S-adenosyl methionine synthase, transketolase, and phenylalanyl t-RNA synthetase beta chain. Four other cDNA sequences had no significant identities with known genes. As revealed by RT-PCR, these genes regulate spine formation during the developmental stage. Our results suggest that PCR-based differential display RT-PCR techniques are a very useful tool for identifying spine-specific genes from carrot seeds.

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.

Synthesis of Oligonucleotides with 3‘-Terminal 5-(3-Acylamidopropargyl)-3‘-amino-2‘,3‘-dideoxyuridine Residues and Their Reactivity in Single-Nucleotide Steps of Chemical Replication

Oligonucleotides with a 3'-terminal 5-alkynyl-3'-amino-2',3'-dideoxyuridine residue were prepared, starting from 2'-deoxyuridine. The optimized route employs a 2',3'-dideoxy-3'-trifluoroacetamido-5-iodouridine 5'-phosphoramidite as building block for DNA synthesis and involves on-support Sonogashira coupling with N-tritylpropargylamine to generate oligonucleotides. The amino group of the propargylamine side chain was acylated to accelerate primer extension reactions involving the 3'-amino group. Three acyl groups were identified that decrease the half-life for DNA-templated extension steps with 7-azabenzotriazole esters of 2'-deoxynucleotides. The residue of 4-pyrenylbutyric acid was found to accelerate primer extension reactions and to render them more selective than those of the control primer. With this substituent, primer extension is also faster than previously measured for three-strand systems involving template, aminoprimer, and a downstream-binding helper oligonucleotide. Fast-reacting primers might become useful for genotyping single nucleotides.

271 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A 35-DAY-OLD CLONED PIG FETUS USING THE ANNEALING CONTROL PRIMER SYSTEM

Somatic cell nuclear transfer in pig has limitations due to the high incidence of fetal failure after embryo transfer to recipients. Reasons for the inefficient cloning are assumed to be due to abnormal and poorly developed placenta. Thus, this study was designed to determine possible genetic causes of neonatal deaths and other related abnormalities. Genes expressed specifically or prominently on Day 35 were identified in cloned pig placenta utilizing PCR technology regulated by annealing control primers (ACPs). The RNA was isolated using Trizol reagent. By utilizing 120 ACPs, 53 expressed sequence tags (ESTs) of genes that are differentially expressed in cloned pig placenta compared with normal placenta were cloned and sequenced. The cloned genes or ESTs exhibited significant sequence similarities to known genes or ESTs of other species. Ten of the total known genes, i.e., pregnancy associated glycoprotein, H19, 60S ribosomal protein L12, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, heme oxygenase 2, granulin precursor, and placenta-expressed transcript protein, were selected and their specific expression levels were confirmed by real-time RT-PCR in the normal and cloned pig placentas in triplicate using beta-actin for determining relative expression. The 60S ribosomal protein L12 and heme oxygenase 2 were highly expressed in the cloned pig placenta, whereas pregnancy associated glycoprotein, H19, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, granulin precursor, and placenta-expressed transcript protein were low or void. Our data suggest that the ACP system effectively identified tissue-specific genes in cloned pig placenta. Furthermore, identified genes would assist in developing insight into the genetic basis of fetal failure and help in resolving low pregnancy rate in the production of cloned pigs.