15025 Correlation of in vitro gene expression analysis with in vivo efficacy

Abstract We have isolated c-Kit+Lin− cells from both human and murine amniotic fluid (AF) and investigated their hematopoietic potential. In vitro, the c-Kit+Lin− population in both species displayed a multilineage hematopoietic potential, as demonstrated by the generation of erythroid, myeloid, and lymphoid cells. In vivo, cells belonging to all 3 hematopoietic lineages were found after primary and secondary transplantation of murine c-Kit+Lin− cells into immunocompromised hosts, thus demonstrating the ability of these cells to self-renew. Gene expression analysis of c-Kit+ cells isolated from murine AF confirmed these results. The presence of cells with similar characteristics in the surrounding amnion indicates the possible origin of AF c-Kit+Lin− cells. This is the first report showing that cells isolated from the AF do have hematopoietic potential; our results support the idea that AF may be a new source of stem cells for therapeutic applications.

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New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors

Applied and Environmental Microbiology ◽

10.1128/aem.01334-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 1

Author(s):

Massimiliano Lucidi ◽

Daniela Visaggio ◽

Elisa Prencipe ◽

Francesco Imperi ◽

Giordano Rampioni ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Multidrug Resistant ◽

Antibiotic Selection ◽

Content Type ◽

Shuttle Vectors ◽

Reporter Systems

ABSTRACT The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA. A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model. IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

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129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab129 ◽

2013 ◽

Vol 25 (1) ◽

pp. 212

Author(s):

G. Machado ◽

A. Ferreira ◽

I. Pivato ◽

A. Fidelis ◽

J. F. Srpicigo ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

T Test ◽

Molecular Profile ◽

Control Group ◽

Fluid Medium ◽

Uterine Flushing

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO2 in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality. Financial support: Embrapa, CNPq, CAPES.

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In vitro nuclear receptor activity and in vivo gene expression analysis in Murray-Darling rainbowfish (Melanotaenia fluviatilis) after short-term exposure to fluoxetine

Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology ◽

10.1016/j.cbpc.2016.05.007 ◽

2016 ◽

Vol 188 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Peter A. Bain ◽

V.S. Basheer ◽

Adrienne Gregg ◽

J.K. Jena ◽

Anu Kumar

Keyword(s):

Gene Expression ◽

Nuclear Receptor ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Receptor Activity ◽

Short Term ◽

Short Term Exposure

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Gene Expression Analysis of Troglitazone Reveals Its Impact on Multiple Pathways in Cell Culture: A Case for In Vitro Platforms Combined with Gene Expression Analysis for Early (Idiosyncratic) Toxicity Screening

International Journal of Toxicology ◽

10.1080/10915810600605690 ◽

2006 ◽

Vol 25 (2) ◽

pp. 85-94 ◽

Cited By ~ 20

Author(s):

Gordon Vansant ◽

Patrick Pezzoli ◽

Robert Saiz ◽

Aaron Birch ◽

Chris Duffy ◽

...

Keyword(s):

Gene Expression ◽

Type 2 Diabetes ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Peroxisome Proliferator ◽

Toxicity Screening ◽

The Impact

Peroxisome proliferator-activated receptor gamma (PPAR γ) agonists of the thiazolidinedione family are used for the treatment of type 2 diabetes mellitus due to their ability to reduce glucose and lipid levels in patients with this disease. Three thiazolidinediones that were approved for treatment are Rezulin (troglitazone), Avandia (rosiglitazone), and Actos (pioglitazone). Troglitazone was withdrawn from the market due to idiosyncratic drug toxicity. Rosiglitazone and pioglitazone are still on the market for the treatment of type 2 diabetes. The authors present data from a gene expression screen that compares the impact these three compounds have in rats, in rat hepatocytes, and in the clone 9 rat liver cell line. The authors monitored the changes in expression in multiple genes, including those related to xenobiotic metabolism, proliferation, DNA damage, oxidative stress, apoptosis, and inflammation. Compared to the other two compounds, troglitazone had a significant impact on many of the pathways monitored in vitro although no major perturbation was detected in vivo. The changes detected predict not only general toxicity but potential mechanisms of toxicity. Based on gene expression analysis, the authors propose there is not just one but multiple ways troglitazone could be toxic, depending on a patient’s environment and genetic makeup, including immune response-related toxicity.

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Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis

Toxicology Letters ◽

10.1016/j.toxlet.2013.06.236 ◽

2013 ◽

Vol 221 (3) ◽

pp. 225-236 ◽

Cited By ~ 11

Author(s):

Anne S. Kienhuis ◽

Alexa P. Vitins ◽

Jeroen L.A. Pennings ◽

Tessa E. Pronk ◽

Ewoud N. Speksnijder ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Cyclosporine A ◽

Gene Expression Analysis ◽

In Vitro Models

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Reciprocal regulation of pancreatic ductal adenocarcinoma growth and molecular subtype by HNF4α and SIX1/4

Gut ◽

10.1136/gutjnl-2020-321316 ◽

2020 ◽

pp. gutjnl-2020-321316

Author(s):

Soledad A Camolotto ◽

Veronika K Belova ◽

Luke Torre-Healy ◽

Jeffery M Vahrenkamp ◽

Kristofer C Berrett ◽

...

Keyword(s):

Gene Expression ◽

Pancreatic Ductal Adenocarcinoma ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Molecular Subtype ◽

Ductal Adenocarcinoma ◽

Reciprocal Regulation ◽

Response To Chemotherapy

ObjectivePancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: classical and basal-like. The classical subtype is characterised by a more favourable prognosis and better response to chemotherapy than the basal-like subtype. The classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor hepatocyte nuclear factor 4 α (HNF4α). The objective of this study is to evaluate the role of HNF4α, SIX4 and SIX1 in regulating the growth and molecular subtype of PDAC.DesignWe manipulate the expression of HNF4α, SIX4 and SIX1 in multiple in vitro and in vivo PDAC models. We determine the consequences of manipulating these genes on PDAC growth, differentiation and molecular subtype using functional assays, gene expression analysis and cross-species comparisons with human datasets.ResultsWe show that HNF4α restrains tumour growth and drives tumour cells toward an epithelial identity. Gene expression analysis of murine models and human tumours shows that HNF4α activates expression of genes associated with the classical subtype. HNF4α also directly represses SIX4 and SIX1, two mesodermal/neuronal lineage specifiers expressed in the basal-like subtype. Finally, SIX4 and SIX1 drive proliferation and regulate differentiation in HNF4α-negative PDAC.ConclusionOur data show that HNF4α regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the classical gene expression programme and repression of SIX4 and SIX1, which may represent novel dependencies of the basal-like subtype.

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