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Find video protocols related to scientific articles indexed in Pubmed.
In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix.
Reprod. Fertil. Dev.
PUBLISHED: 09-13-2014
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Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17?-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17?-oestradiol was observed in culture media from ENC+ compared with ENC- and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen production, consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.
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Network analysis of endogenous gene expression profiles after polyethyleneimine-mediated DNA delivery.
J Gene Med
PUBLISHED: 03-08-2013
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DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI-mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems.
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Microarray analysis of gene expression profiles in cells transfected with nonviral vectors.
Mol. Ther.
PUBLISHED: 08-09-2011
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Inefficient gene delivery is a critical factor limiting the use of nonviral methods in therapeutic applications including gene therapy and tissue engineering. There have been few efforts to understand or engineer the molecular signaling pathways that dictate the efficacy of gene transfer. Microarray analysis was used to determine endogenous gene expression profiles modulated during nonviral gene transfer. Nonviral DNA lipoplexes were delivered to HEK 293T cells. Flow cytometry was used to isolate a population of transfected cells. Expression patterns were compared between transfected and nontransfected samples, which revealed three genes that were significantly upregulated in transfected cells, including RAP1A, a GTPase implicated in integrin-mediated cell adhesion, and HSP70B, a stress-inducible gene that may be important for maintaining cell viability. Furthermore, RAP1A was also significantly upregulated in untransfected cells that were exposed to lipoplexes but that had not expressed the transgene as compared to control, untreated cells. Transfection in the presence of activators of upregulated genes was enhanced, demonstrating the principle of altering endogenous gene expression profiles to enhance transfection. With a greater understanding of signaling pathways involved in gene delivery, more efficient nonviral delivery schemes capitalizing on endogenous factors can be developed to advance therapeutic applications.
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MR elastography monitoring of tissue-engineered constructs.
NMR Biomed
PUBLISHED: 03-08-2011
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The objective of tissue engineering (TE) is to create functional replacements for various tissues; the mechanical properties of these engineered constructs are critical to their function. Several techniques have been developed for the measurement of the mechanical properties of tissues and organs; however, current methods are destructive. The field of TE will benefit immensely if biomechanical models developed by these techniques could be combined with existing imaging modalities to enable noninvasive, dynamic assessment of mechanical properties during tissue growth. Specifically, MR elastography (MRE), which is based on the synchronization of a mechanical actuator with a phase contrast imaging pulse sequence, has the capacity to measure tissue strain generated by sonic cyclic displacement. The captured displacement is presented in shear wave images from which the complex shear moduli can be extracted or simplified by a direct measure, termed the shear stiffness. MRE has been extended to the microscopic scale, combining clinical MRE with high-field magnets, stronger magnetic field gradients and smaller, more sensitive, radiofrequency coils, enabling the interrogation of smaller samples, such as tissue-engineered constructs. The following topics are presented in this article: (i) current mechanical measurement techniques and their limitations in TE; (ii) a description of the MRE system, MRE theory and how it can be applied for the measurement of mechanical properties of tissue-engineered constructs; (iii) a summary of in vitro MRE work for the monitoring of osteogenic and adipogenic tissues originating from human adult mesenchymal stem cells (MSCs); (iv) preliminary in vivo studies of MRE of tissues originating from mouse MSCs implanted subcutaneously in immunodeficient mice with an emphasis on in vivo MRE challenges; (v) future directions to resolve current issues with in vivo MRE in the context of how to improve the future role of MRE in TE.
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Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion.
Yeast
PUBLISHED: 05-30-2010
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Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins. Both proteins showed conserved glutamine (Q) and aspartic acid (D) in the repeated sequences, indicating a possible alkali-sensitive ?1,3-glucan ester linkage. The fusion constructs of PpPir1-2p and enhanced green fluorescent protein (EGFP) were developed for yeast cell surface display. The immunofluorescence assay showed uniform localization of EGFP protein on the P. pastoris cell surface in all fusion constructs. Furthermore, new vectors were developed for recombinant protein secretion in P. pastoris, utilizing the pre-pro signal of PpPir1p protein. Efficient processing of the signal sequence was observed from EGFP and human ?1-antitrypsin (AAT) fusion constructs and recombinant protein secretion was obtained in the culture supernatant.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.