434 REPLICATION COMPETENT LENTIVIRUS (RCL) ANALYSIS IN RECIPIENT ANIMALS OF TRANSGENIC EMBRYOS PRODUCED BY LENTIVIRAL TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 374
Author(s):  
K. Tessanne ◽  
J. Yao ◽  
K. Cornetta ◽  
M. Westhusin ◽  
T. Spencer ◽  
...  
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Lentiviral Vectors ◽  
Transgenic Animal ◽  
Animal Production ◽  
Qrt Pcr ◽  
Fetal Fibroblasts ◽  
Transgenic Embryos

Lentiviral vectors have become a useful tool for gene therapies and the expression of small hairpin (sh)RNAs to target genes both in vitro and in vivo. This is due primarily to their ability to integrate transgenes into both dividing and nondividing cells, as well as the lack of silencing in the germ cell line. However, the retroviral basis for these recombinant, replication-incompetent viruses has prompted investigation into their safety for use in therapeutics and transgenic animal production. Concerns are that recombination with wild-type viruses or endogenous retroviral elements may allow the integrated provirus genome to become replication competent. In order to investigate this, transgenic embryos produced by lentiviral-mediated gene transfer were transferred into recipient animals. The lentiviral plasmids used in this experiment contained a self-inactivating 3′ untranslated region as well as a green fluorescent protein (GFP) reporter gene (Miyoshi et al. 1998 J.Virol. 72, 8150-8157). Recombinant lentivirus was produced through cotransfection of HEK293T cells with the lentiviral transfer plasmid as well as a packaging plasmid and a plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), which was used to pseudotype viral particles. Two methods were used for production of transgenic embryos. The first was lentiviral transduction of bovine fetal fibroblasts followed by somatic cell nuclear transfer. The second was incubation of IVP hatched ovine blastocysts in culture medium containing infectious recombinant lentiviral particles. Recipients were then sacrificed and analyzed for the presence of replication competent lentivirus (RCL). Tissues collected from each recipient included blood, lung, lymph node, kidney, liver, mammary gland, ovary, skeletal muscle, spleen, and uterus. In addition, when available, fetal and placental samples were collected. Analyses for RCL included a serum ELISA test for presence of the p24 HIV antigen as well as real-time quantitative PCR (qRT-PCR) on genomic DNA for the presence of VSV-G. To date, a total of 13 recipients including both sheep and cattle have been analyzed. All animals had p24 titers below the level of detection for the assay (<12.5 pg mL-1). Additionally, the tissues mentioned above have been analyzed by qRT-PCR for 6 of the 13 recipients so far, and all have been negative for VSV-G as determined by comparison with positive and negative control samples. Additional collections and analysis are ongoing. A lack of detection of RCL in these animals will build confidence in the use of lentiviral vectors in transgenic animal production and will lend support for their safety in both animal and human therapies.

scholarly journals MicroRNA-34a Attenuates Paclitaxel Resistance in Prostate Cancer Cells via Direct Suppression of JAG1/Notch1 Axis

2018 ◽  
Vol 50 (1) ◽  
pp. 261-276 ◽  
Author(s):  
Xiaobing Liu ◽  
Xing Luo ◽  
Yuqi Wu ◽  
Ding Xia ◽  
Wei Chen ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Growth ◽  
Western Blot ◽  
Target Genes ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Qrt Pcr

Background/Aims: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance. Methods: We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay. Results: miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis. Conclusion: Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.

Ultrastructure of vitrified rabbit transgenic embryos

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 558-564 ◽  
Author(s):  
P. Chrenek ◽  
A.V. Makarevich ◽  
M. Popelková ◽  
J. Schlarmannová ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Ultrastructural Level ◽  
Membrane Structures ◽  
Egfp Gene ◽  
Cell Structures ◽  
Morula Stage ◽  
Transgenic Embryos ◽  
Green Fluorescent

SummaryThe aim of the study was to determine the viability of rabbit transgenic (enhanced green fluorescent protein (EGFP)-positive) embryos cultured in vitro and compare with gene-microinjected (Mi) non-transgenic (EGFP-negative) embryos following vitrification. Non-microinjected and non-vitrified embryos were used as the control. Morphological signs of injury to embryo organelles were determined at the ultrastructural level using transmission electron microscopy (TEM). Morphometric evaluation was performed on cellular organelles using microphotographs obtained by TEM. Intact and Mi embryos recovered from in vivo fertilized eggs at 19–20 hours post coitum (hpc) were cultured for up to 72 hpc (morula stage), evaluated for the EGFP gene integration and then vitrified in 0.25 ml insemination straws in modified EFS (40% ethylene glycol + 18% Ficoll 70 + 0.3 M sucrose) vitrification solution. After 1–3 days the embryos were devitrified, a representative selection of embryos was analyzed by TEM and the remaining embryos were subjected to additional in vitro culture. Observations by TEM showed that the vitrified/warmed EGFP-positive and EGFP-negative embryos had a slight accumulation of cellular debris and lipid droplets compared with the control intact embryos. More severe changes were detected in the membrane structures of the treated embryos, mostly in the cytoplasmic envelope, trophoblastic microvilli, junctional contacts and mitochondria. We suggest that the higher proportion of deteriorated cell structures and organelles in the treated embryos may be due to the vitrification process rather than to mechanical violation (the gene-microinjection procedure), as a detailed inspection of ultrastructure revealed that most damage occurred in the cell membrane structures.

scholarly journals A Novel System for Monitoring in vivo Cell Signaling Pathways Involved in Early Embryonic Patterning

2021 ◽  
Author(s):  
◽  
Louise Rooney
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Cell Signalling ◽  
Original System ◽  
Signal Propagation ◽  
Mouse Embryonic Fibroblast ◽  
Embryonic Patterning ◽  
Transgenic Embryos

<p>Early developmental events, such as the arrangement of the head-tail axis, are fundamentally driven by cell signalling cascades. Such incidents are regulated in a highly complex manner by promoters and inhibitors at many levels of the cascade. This complexity makes it difficult to understand where and when certain signalling occurs, and what effects additional factors have on the signalling system. Nodal signalling, executed by intracellular Smad2/3 signal propagation, is thought to induce the anterior-posterior and head-tail patterning of the early mouse embryo. Target gene outputs of this signalling are fine-tuned by a vast array of modulators; TGBβ co-receptors, extracellular ligand and receptor inhibitors, DNA binding cofactors, and intracellular enhancers and inhibitors. The endogenous target genes of this system cannot be used as a measure of signalling as they themselves feedback on the original system and others, creating diverse signals. In this body of work, we have distilled the Nodal signalling cascade to a single variable by creating a fluorescent genetic reporter to semi-quantitatively measure Smad signalling during early embryonic development. Reporter constructs contain Smad binding elements, a minimal promoter and fluorescent protein elements. Various sensitivity Smad binding elements were created to respond to different thresholds of signalling. Fluorescent microscopy and flow cytometry were used to verify responsiveness of reporter constructs, tested first in a mouse embryonic fibroblast line and subsequently in transgenic embryos. This study will provide an understanding of how extracellular cues dictate gene expression during early embryonic formation. The knowledge acquired from this work may have implications in dairy cattle and human fertility.</p>

scholarly journals A Novel System for Monitoring in vivo Cell Signaling Pathways Involved in Early Embryonic Patterning

2021 ◽  
Author(s):  
◽  
Louise Rooney
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Cell Signalling ◽  
Original System ◽  
Signal Propagation ◽  
Mouse Embryonic Fibroblast ◽  
Embryonic Patterning ◽  
Transgenic Embryos

<p>Early developmental events, such as the arrangement of the head-tail axis, are fundamentally driven by cell signalling cascades. Such incidents are regulated in a highly complex manner by promoters and inhibitors at many levels of the cascade. This complexity makes it difficult to understand where and when certain signalling occurs, and what effects additional factors have on the signalling system. Nodal signalling, executed by intracellular Smad2/3 signal propagation, is thought to induce the anterior-posterior and head-tail patterning of the early mouse embryo. Target gene outputs of this signalling are fine-tuned by a vast array of modulators; TGBβ co-receptors, extracellular ligand and receptor inhibitors, DNA binding cofactors, and intracellular enhancers and inhibitors. The endogenous target genes of this system cannot be used as a measure of signalling as they themselves feedback on the original system and others, creating diverse signals. In this body of work, we have distilled the Nodal signalling cascade to a single variable by creating a fluorescent genetic reporter to semi-quantitatively measure Smad signalling during early embryonic development. Reporter constructs contain Smad binding elements, a minimal promoter and fluorescent protein elements. Various sensitivity Smad binding elements were created to respond to different thresholds of signalling. Fluorescent microscopy and flow cytometry were used to verify responsiveness of reporter constructs, tested first in a mouse embryonic fibroblast line and subsequently in transgenic embryos. This study will provide an understanding of how extracellular cues dictate gene expression during early embryonic formation. The knowledge acquired from this work may have implications in dairy cattle and human fertility.</p>

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 339-346 ◽  
Author(s):  
Liangxue Lai ◽  
Qingyuan Sun ◽  
Guangming Wu ◽  
Clifton N. Murphy ◽  
Birgit Kühholzer ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Pronuclear Formation

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8±17.3% vs 28.5±3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0±7.0% vs 63.3±12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0±11.6% vs 4.6±4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Junjie Cen ◽  
Yanping Liang ◽  
Yong Huang ◽  
Yihui Pan ◽  
Guannan Shu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Target Genes ◽  
Expression Patterns ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

scholarly journals TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Rachael Vaubel ◽  
Ann Mladek ◽  
Yu Zhao ◽  
Shiv K Gupta ◽  
Minjee Kim ◽  
...  
Keyword(s):  
Target Genes ◽  
Xenograft Model ◽  
Culture Conditions ◽  
Patient Derived Xenograft ◽  
Fold Induction ◽  
Extended Survival ◽  
Mdm2 Amplification ◽  
Mdm2 Inhibitor

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 &gt;3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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