PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection

Objective: The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection

Materials and Methods: In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer [SCNT] and sperm mediated gene transfer [SMGT] approaches

Results: PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage [attP] sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction [PCR] and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique

Conclusion: The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein [EGFP] positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection [ICSI] method

Expression profile of developmentally important genes in pre- and peri-implantation goat embryos produced in vitro

Background: Little is understood about the regulation of gene expression during early goat embryo development. This study investigated the expression profile of 19 genes, known to be critical for early embryo development in mouse and human, at five different stages of goat in vitro embryo development [oocyte, 8-16 cell, morula, day-7 blastocyst, and day 14 blastocyst]

Materials and Methods: In this experimental study, stage-specific profiling using real time-quantitative polymerase chain reaction [RT-qPCR] revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on their respective mRNA profiles

Results: The gradual pattern of reduction in the maternally stored transcripts without renewal thereafter [cluster-1: Lifr1, Bmpr1, Alk4, Id3, Ctnnb, Akt, Oct4, Rex1, Erk1, Smad1 and 5] implies that their protein products are essential during early cleavages when the goat embryo is silent and reliant to the maternal legacy of mRNA. The potential importance of transcription augment at day-3 [cluster-2: Fzd, c-Myc, Cdc25a, Sox2] or day-14 [cluster-3: Fgfr4, Nanog] suggests that they are nascent embryonic mRNAs which intimately involved in the overriding of MET or regulation of blastocyst formation, respectively. The observation of two expression peaks at both day-3 and day-14 [cluster-4: Gata4, Cdx2] would imply their potential importance during these two critical stages of pre-and peri-implantation development

Conclusion: Evolutionary comparison revealed that the selected subset of genes has been rewired in goat and human/goat similarity is greater than the mouse/goat or bovine/goat similarities. The developed profiles provide a resource for comprehensive understanding of goat preimplantation development and pluripotent stem cell engineering as well

Stage-specific profiling of transforming growth factor-beta, fibroblast growth factor and wingless-int signaling pathways during early embryo development in the goat

Objective: This research intends to unravel the temporal expression profiles of genes involved in three developmentally important signaling pathways [transforming growth factor-beta [TGF-beta], fibroblast growth factor [FGF] and wingless/int [WNT]] during pre- and peri-implantation goat embryo development

Materials and Methods: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the downstream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 [D7] blastocysts and day-14 [D14] blastocysts] were separately prepared in triplicate for comparative real-time reverse transcriptase polymerase chain reaction [RT-PCR] using the selected gene sets

Results: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-beta, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-beta, FGF and WNT components with renewal thereafter

Conclusion: The results suggested that TGF-beta, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of pre- and peri-implantation goat embryo development

An Ontology-Based GIS for Genomic Data Management of Rumen Microbes

During recent years, there has been exponential growth in biological information. With the emergence of large datasets in biology, life scientists are encountering bottlenecks in handling the biological data. This study presents an integrated geographic information system (GIS)-ontology application for handling microbial genome data. The application uses a linear referencing technique as one of the GIS functionalities to represent genes as linear events on the genome layer, where users can define/change the attributes of genes in an event table and interactively see the gene events on a genome layer. Our application adopted ontology to portray and store genomic data in a semantic framework, which facilitates data-sharing among biology domains, applications, and experts. The application was developed in two steps. In the first step, the genome annotated data were prepared and stored in a MySQL database. The second step involved the connection of the database to both ArcGIS and Protege as the GIS engine and ontology platform, respectively. We have designed this application specifically to manage the genome-annotated data of rumen microbial populations. Such a GIS-ontology application offers powerful capabilities for visualizing, managing, reusing, sharing, and querying genome-related data.

Identification of a specific pseudo attP site for phage PhiC31 integrase in bovine genome

PhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene. Identification of a novel pseudo attP site. Genomic DNA was extracted from primary bovine fetal fibroblast cells, which were stably trans-fected with EGFP and phiC31 integrase cDNAs carrying vectors. An inverse PCR was carried out for production of mini-circle DNAs and followed by sequencing. A new specific pseudo attP site termed BF5 was identified in bovine genome. This site is located in an intergenic AT rich region on chromosome 5 with similar features of other mammalian attP pseudo sites. Furthermore, direct sequencing of generated attL site confirmed that site-specific transgene recombination was occurred at this site. This finding confirmed that phiC31 integrase could be feasible for production of transgenic animals for biotechnological applications

Quantiation of IL-4, IL-10 and IFN-gamma genes expression after immunization of mice with CFP-10 and ESAT-6 containing vectors

Tuberculosis is a disease with high morbidity, caused mainly by Mycobaterium tuberculosis [M.tb.]. DNA vaccines show a promising future due to their unique advantages over conventional methods. The early-secreted antigen target [ESAT]-6 and culture filtrate protein [CFP]-10 of M.tb. antigens have been identified as vaccine candidates against Mycobacteria and used as subunit vaccines, DNA or protein, in different studies. To investigate the potential of pcDNA3.1+ plasmid containing CFP-10 and ESAT-6 genes in induction of local immune responses after intramuscular injection in BALB/c mice. pcDNA 3.1+ CFP-10 and pcDNA3.1+ ESAT-6 plasmids were prepared and defined groups of mice were injected intramuscularly with the plasmids both separately and in combination. The RNA was extracted from muscles after one month and cDNA was made using RT-PCR. The expressions of IL-4, IL-10 and IFN-gamma genes cytokines were evaluated using comparative real time PCR. Expression of IL-4 and IL-10 increased in the injection site of the mice groups which received plasmids encoding ESAT-6 and CFP-10 individually or together. More than 10-fold increase in IFN-gamma expression was found in samples taken from mice groups inoculated by plasmids encoding ESAT-6 and CFP-10 individually or together. pcDNA 3.1+ESAT-6 and pcDNA3.1+CFP-10 plasmids can increase the expression of IFN-gamma in mice after immunization.

Cloning, expression, and in vitro functional activity assay of phiC31 integrase cDNA in Escherichia coli

The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 [DE3]. Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] of the purified phiC31 integrase confirmed the size of protein [70 kDa]. Finally, the functionality of purified phiC31 integrase was verified. The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration