136 FUNCTIONAL ANALYSIS OF RHOPHILIN-2 GENE IN PRE-IMPLANTATION MOUSE EMBRYO

2006 ◽  
Vol 18 (2) ◽  
pp. 176
Author(s):  
T. Matsuoka ◽  
Y. Sono ◽  
K. Matsumoto ◽  
T. Amano ◽  
S. Mizuno ◽  
...  
Keyword(s):  
Real Time ◽  
Hybrid System ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Subsequent Development ◽  
Taqman Probe ◽  
Cell Stage ◽  
Transcript Levels ◽  
Cytoskeletal Organization ◽  
G2 Phase

Zygotic gene activation (ZGA), which starts at the G2 phase at the 1-cell stage (Latham 1999), promotes the reprogramming of gene expression and is critical for the subsequent development of pre-implantation embryos. We have investigated the molecule function of many gene clusters, DD clones obtained by Differential-Display assays for ovulated eggs at the M II stage, and 1-cell embryos at the G2 phase. The differential expression of rhophilin-2 shown in DD assays was also confirmed by 3 independent real-time PCR analyses (P < 0.05). For these reasons, in this study, we focused on the rhophilin-2 gene, which regulates cytoskeletal organization (Peck et al. 2002). At first, we identified a protein that interacts with the Rhophilin-2 protein by a yeast 2-hybrid system. To confirm the interaction between Rhophilin-2 and the putative protein obtained by a yeast two-hybrid system, we used a co-immunoprecipitaion assay. We also investigated the expression profiles of rhophilin-2 and the transcripts of the identified protein in ovary and pre-implantation embryos using real-time PCR and immunofluorescence (IF) analysis. The ICR mice at 48 h after PMSG priming were primed with hCG, and ovaries were collected at 7 h after hCG priming. Pre-implantation embryos were collected at 1-cell, 2-cell, and 4-cell stages, and cDNA was produced by mRNA isolated from 10 oocytes or embryos in each group and was subjected to real-time PCR using a TaqMan Probe system (ABI). Sectioned ovaries and pre-implantation embryos were analyzed by IF analysis using antibody of Rhophilin-2 and the identified protein. This is the first report that GABA receptor-association protein (GABARAP) was identified as a protein that interacts with Rhophilin-2, as a result of using the yeast 2-hybrid system and subsequent co-immunoprecipitation assay. After fertilization, transcript levels of rhophilin-2 significantly decreased from the 1-cell stage to the 2-cell stage (P < 0.05), but transcript levels of GABARAP significantly increased from the 1-cell stage to the 2-cell stage (P < 0.05). The IF analysis revealed localization of Rhophilin-2 and GABARAP at the nucleolus of all follicle stage in the ovary. Moreover, Rhophiln-2 and GABARAP were found to be localized on the microtubules of 1-cell and 2-cell embryos, but no signal of Rhophilin-2 was detected in 4-cell embryos. These results suggest that Rhophilin-2 protein regulates the cytoskeletal organization in 1-cell to 2-cell embryos and is involved in the molecular mechanism of cell division by coupling with GABARAP. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

281 EXPRESSION PROFILE AND KNOCKDOWN ANALYSIS OF A FUNCTIONALLY UNKNOWN DD2-2 GENE IN MOUSE PRE-IMPLANTATION EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
S. Shin ◽  
K. Matsumoto ◽  
T. Amano ◽  
K. Saeki ◽  
Y. Hosoi ◽  
...  
Keyword(s):  
Real Time ◽  
Expression Profile ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Gene Clusters ◽  
Egfp Expression ◽  
Nucleotide Sequence Analysis ◽  
Cell Stage ◽  
Unknown Gene ◽  
G2 Phase

Zygotic gene activation (ZGA) starts at the G2 phase at the 1-cell stage in the mouse. However, the molecular mechanism of ZGA has not been completely elucidated. We have investigated the molecular functions of many gene clusters, DD clones obtained by differential display assays for ovulated eggs at the M II stage and 1-cell stage embryos at the G2 phase. As a result, we have identified a functionally unknown gene, whose sequence did not match a known transcript in the gene bank DD2-2 gene. Here, we report the expression profile and knockdown analysis of the DD2-2 gene in mouse pre-implantation embryos. Nucleotide sequence analysis of the DD2-2 cDNA revealed that the open reading frame of 1056 bp encodes a protein of 351 amino acids with a predicted molecular mass of 41.5 kDa. The deduced amino acid sequence indicated that DD2-2 protein might be a soluble protein without a signal peptide. We first investigated the expression profiles of DD2-2 in pre-implantation embryos by quantitative real-time PCR using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). To investigate the effect of knockdown of the DD2-2 gene on the development of pre-implantation embryos, we injected pβ-act/antisenseDD2-2/IRES/EGFP into male pronuclei of embryos at 7 to 9 h after insemination (hpi) and observed the development of embryos that showed EGFP expression at 24 hpi. Real-time PCR analysis of pre-implantation embryos showed that maternal DD2-2 mRNA at a low level significantly increased up to the early 2-cell stage, and significantly decreased by the 4-cell stage and later, suggesting that DD2-2 gene specifically expresses at major ZGA. In the knockdown analysis, EGFP-positive embryos with pβ-act/antisenseDD2-2/IRES/EGFP showed a lower rate of development to the 4-cell stage and later, compared with that of EGFP-positive embryos with pβ-act/luc+/IRES/EGFP [72% (94/130) vs. 54% (71/131); P &lt; 0.05], indicating that the knockdown of DD2-2 by antisense RNA resulted in a inhibition of pre-implantation development. In conclusion, the DD2-2 gene, a functionally unknown gene, may play an important role in pre-implantation development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

118 EXPRESSION PROFILES OF CIRCADIAN CLOCK GENES IN MOUSE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 167
Author(s):  
T. Amano ◽  
A. Matsushita ◽  
R. Kakegawa ◽  
K. Matsumoto ◽  
K. Saeki ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Real Time ◽  
Nuclear Localization ◽  
Real Time Pcr ◽  
Clock Genes ◽  
Expression Profiles ◽  
Germinal Vesicle ◽  
Cell Stage ◽  
Mrna Extraction ◽  
Circadian Clock Genes

Matsuo et al. reported that circadian clock genes regulate the timing of cell division in mouse regenerating liver cells (2003). Their results suggested the importance of circadian clock genes for organs or tissues for which functions are characterized by cell division, such as pre-implantation embryos. To obtain basic information on the molecular functions of circadian clock genes in pre-implantation embryos, we investigated the expression profiles of transcripts and proteins of some circadian clock genes, clock, bmal1, cry1, and per2, in mouse germinal vesicle oocytes (GV), MII oocytes (MII), and pre-implantation embryos using real-time PCR and immunocytochemistry (ICC). Germinal vesicle oocytes were collected from ICR females at 48 h after PMSG priming. The mouse at 48 h after PMSG priming was primed with hCG, and MII were collected at 15 h after hCG priming. The pre-implantation embryos were collected at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after insemination, and they corresponded to early 1-cell, late 1-cell, early 2-cell, late 2-cell, 4-cell, 8-cell, early morula, late morula, and blastocyst stages, respectively. cDNA was produced by mRNA isolated from 20 oocytes or embryos using oligo dT and was subjected to real-time PCR using a TaqMan Probe system (ABI). Three sets of 20 oocytes or embryos at each developmental stage were applied to mRNA extraction and real-time PCR analysis to ensure equal mRNA extraction efficiency between samples. The level of mRNA of each clock gene contained in 3 samples from each developmental stage was almost the same. Statistical analysis of the transcripts of each gene were done by ANOVA. Germinal vesicles, MII and embryos collected at each time point were subjected to ICC using antibodies of CLOCK, BMAL1, CRY1, and PER2. The oocytes or embryos treated with only secondary antibody did not produce any signal. All of the examined genes except per2 were expressed in oocytes and pre-implantation embryos. The transcript level of clock, bmal1, and cry1 in MII were significantly lower than those in GV (P < 0.05). After fertilization, transcript levels of clock, bmal1, and cry1 significantly decreased from early 1-cell stage to late 2-cell stage (P < 0.05). These decreased transcript levels were maintained until the blastocyst stage after the late 2-cell stage. Immunocytochemistry analysis showed the nuclear localization of CLOCK and BMAL1 in early and late 2-cell embryos and of CRY1 in early 2-cell embryos but no signals of PER2 in oocytes or pre-implantation embryos. Because mouse oocytes and 1- to 2-cell embryos are transcriptionally inert, the abundant transcripts of clock, bmal1, and cry1 in these stages seemed to indicate that they were synthesized and stored during the oocyte growth phase. Moreover, the nuclear localization of CLOCK, BMAL1, and CRY1 in the oocytes and 1- to 2-cell stage embryos suggested that some clock genes were translated and worked for oocyte maturation and early embryogenesis. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

103 GENE EXPRESSION PROFILES OF VITRIFIED MOUSE EMBRYOS DETECTED BY MICROARRAYS

2006 ◽  
Vol 18 (2) ◽  
pp. 160
Author(s):  
S. Mamo ◽  
Sz. Bodo ◽  
Z. Polgar ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Differentially Expressed ◽  
Analysis Tool ◽  
Cell Stage ◽  
Base Medium ◽  
Independent Analysis

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to examine the transcript variations and identify genes most affected by the treatment. For this, 8-cell-stage embryos were collected from female ICR mice mated with ICR males. The embryos were washed with CZB-HEPES base medium and suspended briefly in equilibrium medium consisting of 4% ethylene glycol (EG) in base medium at room temperature. Following equilibration, the embryos were vitrified in a 35% EG, 0.4 M trehalose, 5% polyvinylpyrrolidone (PVP) solution by means of a solid-surface vitrification (SSV) technique as described earlier (Dinnyes 2000 Biol. Reprod. 63, 513-518). Then 40 embryos each from the control and the vitrified/warmed groups were cultured in CZB medium for 3 h. Total RNAs were extracted from cultured embryos in each group using TRIzol (Invitrogen, Bio-Science, Ltd., Budapest, Hungary), following the manufacturer's instructions. Two rounds of amplification were employed to produce labeled RNA, using low input RNA amplification kit (Agilent Technologies, Kromat, Ltd., Budapest, Hungary) procedures with modifications. Three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22K oligonucleotide slides with subsequent analysis of the results. Moreover, as an independent analysis tool, real time PCR was used with eight designed primers. All of the vitrified embryos were recovered after warming with no morphological signs of cryodamage and used for analysis. The two rounds of amplification yielded 15-16 �g of cRNA. The analysis of repeated hybridizations by Rosetta luminator software (Agilent) showed 20 183 genes and expressed sequence tags (ESTs) that passed the selection criteria and were identified as common signatures in all of the slides. Unsupervised analysis of the gene expression data identified a total of 631 differentially expressed (P < 0.01) genes. However, to support the reliability of the results, only those variations above 1.5 fold differences were considered as significant in the final analysis. Therefore, with this stringent criterion 183 genes were differentially expressed (P < 0.01), of which 109 were up-regulated and the remainder down-regulated. Although genes have multiple and overlapping functions, most of the differentially expressed genes were functionally classified into various physiological categories. These include stress response (8), apoptosis related (6), metabolism (51), temperature response (4), and transcription regulation (15). Moreover, the independent analysis with real time PCR and unamplified samples verified the results of microarray. Thus, based on confirmation of the results by an independent analysis and support by the previous studies for some of the genes, it is possible to conclude that the expression patterns reflect the true biological image of embryos after vitrification, with most effects on stress- and cell metabolism-related genes. This work was supported by EU FP6 (MEXT-CT-2003-59582), Wellcome Trust Foundation (Grant No. 070246), and National Office of Research and Technology (NKTH) (#BIO-00017/2002, #BIO-00086/2002).

189 TRANSCRIPTIONAL PROFILING OF HISTONE-MODIFYING GENES DURING BOVINE PRE-IMPLANTATION EMBRYO DEVELOPMENT IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 193
Author(s):  
G. D. Linger ◽  
C. L. Bormann ◽  
M. D. Peoples ◽  
M. C. Golding ◽  
C. R. Long
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Geometric Mean ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Morula Stage

The proper removal of gametic epigenetic marks and coordinated re-establishment of the epigenome is critical to mammalian embryonic development. This global reprogramming of the embryonic genome includes fluctuations in both DNA methylation and histone modifications that are necessary to control chromatin structure and thus gene expression. In the bovine model, epigenetic changes occur from fertilization through blastocyst stages; in particular, and concurrent with the maternal-embryonic transition, de novo DNA methylation begins at the 8-cell stage. In order to understand which factors might be playing key roles in this epigenetic process, we used quantitative real-time PCR to characterize the temporal expression profiles of several genes involved in DNA and/or histone methylation: G9a, SetB1, Suv39h1, Suv420h1, SmyD3, Suz12, and LSH. Bovine ova and embryos were produced via in vitro maturation, fertilization, and culture from multiple pools of ova. Groups of 12–25 bovine ova or embryos, pooled at the 2-, 4 to 7-, mid 8-, late 8-, 12 to 16-cell, morula, and blastocyst stages, were washed twice through 1X PBS and stored in RNA lysis buffer at –80°C until further use. RNA was isolated from each sample using the RNeasy® Mini kit (Qiagen, Valencia, CA, USA), optimized for isolating RNA from single embryos, and treated to remove any contaminating genomic DNA. cDNA was generated with iScript™ reverse transcriptase (Bio-Rad Laboratories, Hercules, CA, USA) and diluted 1:10 with RNase/DNase-free water for further use in real-time PCR. Relative gene expression from each RNA sample was calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA, USA) adjusted for individual PCR efficiencies (Bustin 2003) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA) in order to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). G9a and SetB1, both lysine-specific methyltransferases, were expressed at their highest levels in the metaphase II (MII) oocyte and 2-cell stage, before expression decreased gradually to basal levels by the morula and blastocyst stages. Suv39h1, Suv420h1, and SmyD3, also lysine-specific methyltransferases, all shared a similar pattern of expression: transcript levels were fairly high in the MII oocyte, increased at the 2-cell stage, then gradually dropped off around the 8–16-cell stage to basal levels by the morula stage. Interestingly, Suz12 and LSH both showed low expression from the MII oocyte until the 4 to 7-cell stage, increased dramatically at the 8-cell stage, then decreased again by the morula stage. Suz12 is a member of several Polycomb group complexes (PRCs); LSH associates with PRC-mediated gene silencing as well as DNMT3a and 3b. These data suggest that Suz12 and LSH may be implicated in bovine embryonic genome activation, while the latter genes are active during earlier cleavage events. Ongoing studies will evaluate the role of each of these epigenetic modifiers in bovine pre-implantation embryos by selective silencing via RNA interference.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Evaluation of droplet digital PCR for the detection of black canker disease in tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Li Wang ◽  
Tian Qian ◽  
Pei Zhou ◽  
Wenjun Zhao ◽  
Xianchao Sun
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Detection Threshold ◽  
Digital Pcr ◽  
Taqman Probe ◽  
Tomato Seed ◽  
Canker Disease ◽  
Probe Set

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

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