scholarly journals HPV16-E6 Oncoprotein Activates TGF-βand Wnt/β-Catenin Pathways in the Epithelium-Mesenchymal Transition of Cataracts in a Transgenic Mouse Model

2018 ◽  
Vol 2018 ◽  
pp. 1-17
Author(s):  
Genaro Rodríguez-Uribe ◽  
Nicolas Serafín-Higuera ◽  
Gabriela Damian-Morales ◽  
Enoc Mariano Cortés-Malagón ◽  
Vicky García-Hernández ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Transgenic Mouse Model ◽  
Rt Pcr ◽  
Hpv16 E6 ◽  
Mesenchymal Transition ◽  
Protein Levels ◽  
E6 Oncoprotein ◽  
Signaling Activation ◽  
Smad7 Protein

Objective. This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-βand Wnt/β-catenin signaling activation.Materials and Methods. Cataracts of K14E6 mice were analysed histologically; and components of TGF-βand Wnt/β-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor.Results. The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses’ cell morphology were due to actin filaments reorganisation, activation of TGF-βand Wnt/β-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels.Conclusion. The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-βand Wnt/β-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

A transgenic mouse model for the ductal carcinoma in situ (DCIS) of the mammary gland

Oncogene ◽  
2000 ◽  
Vol 19 (8) ◽  
pp. 1028-1037 ◽  
Author(s):  
Christine Schulze-Garg ◽  
Jürgen Löhler ◽  
Andreas Gocht ◽  
Wolfgang Deppert
Keyword(s):  
Mammary Gland ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Ductal Carcinoma In Situ ◽  
Carcinoma In Situ ◽  
Ductal Carcinoma ◽  
Transgenic Mouse Model

The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFβ pathway in the epidermis of the K14E6 transgenic mouse

2015 ◽  
Vol 24 (6) ◽  
pp. 430-435 ◽  
Author(s):  
Marco Popoca-Cuaya ◽  
Jose Diaz-Chavez ◽  
Jesus Hernandez-Monge ◽  
Elizabeth Alvarez-Rios ◽  
Paul F. Lambert ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Transgenic Mouse ◽  
Hpv16 E6 ◽  
Uvb Irradiation ◽  
E6 Oncoprotein ◽  
Tgfβ Pathway

scholarly journals A transgenic mouse model ubiquitously overexpressing Dnmt1s mRNA lacks increased protein levels

2014 ◽  
Author(s):  
Christian Grosser ◽  
Lothar Vassen ◽  
Bernhard Horsthemke ◽  
Nicholas Wagner
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Transgenic Mouse Model ◽  
Protein Levels ◽  
Established Role ◽  
Cag Promoter ◽  
Egfp Fluorescence

Several types of cancer are characterized by global hypomethylation accompanied by regional hypermethylation and overexpression of DNA methyltransferase (cytosine-5) 1 (Dnmt1). In addition to the established role of Dnmt1 as maintenance methyltransferase, it has been suggested that Dnmt1 might also methylate certain target sites de novo. We created a transgenic mouse model to investigate whether the overexpression of the somatic form of Dnmt1, Dnmt1s, is sufficient to cause erroneous methylation and disease. Because ubiquitous Dnmt1 overexpression has been reported to be embryonic lethal, we designed a CAG promoter-driven Cre-loxP conditional transgene containing a floxed EGFP sequence followed by the Dnmt1s coding sequence. The EGFP sequence is excised and transgenic Dnmt1s expression is activated at specific time points or in specific tissues depending on the Cre deleter strain used for cross-ins. Pronucleus injections with the Dnmt1s transgene construct resulted in six founder lines as verified by PCR, Southern blot and EGFP fluorescence. We performed cross-ins with a CMV-Cre deleter strain to clarify if ubiquitous overexpression of Dnmt1s alone causes the previously observed embryonic lethality. Unexpectedly, these cross-ins yielded viable recombined offspring that ubiquitously overexpressed Dnmt1 mRNA at tissue-dependent levels of up to 229-fold. However, the recombined offspring did not significantly overexpress Dnmt1 protein and showed no apparent signs of disease or pathological phenotype. Here, we describe the establishment of our Dnmt1s-transgenic mouse model and propose possibilities for the absence of transgenic protein.

scholarly journals A transgenic mouse model ubiquitously overexpressing Dnmt1s mRNA lacks increased protein levels

2014 ◽  
Author(s):  
Christian Grosser ◽  
Lothar Vassen ◽  
Bernhard Horsthemke ◽  
Nicholas Wagner
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Transgenic Mouse Model ◽  
Protein Levels ◽  
Established Role ◽  
Cag Promoter ◽  
Egfp Fluorescence

Several types of cancer are characterized by global hypomethylation accompanied by regional hypermethylation and overexpression of DNA methyltransferase (cytosine-5) 1 (Dnmt1). In addition to the established role of Dnmt1 as maintenance methyltransferase, it has been suggested that Dnmt1 might also methylate certain target sites de novo. We created a transgenic mouse model to investigate whether the overexpression of the somatic form of Dnmt1, Dnmt1s, is sufficient to cause erroneous methylation and disease. Because ubiquitous Dnmt1 overexpression has been reported to be embryonic lethal, we designed a CAG promoter-driven Cre-loxP conditional transgene containing a floxed EGFP sequence followed by the Dnmt1s coding sequence. The EGFP sequence is excised and transgenic Dnmt1s expression is activated at specific time points or in specific tissues depending on the Cre deleter strain used for cross-ins. Pronucleus injections with the Dnmt1s transgene construct resulted in six founder lines as verified by PCR, Southern blot and EGFP fluorescence. We performed cross-ins with a CMV-Cre deleter strain to clarify if ubiquitous overexpression of Dnmt1s alone causes the previously observed embryonic lethality. Unexpectedly, these cross-ins yielded viable recombined offspring that ubiquitously overexpressed Dnmt1 mRNA at tissue-dependent levels of up to 229-fold. However, the recombined offspring did not significantly overexpress Dnmt1 protein and showed no apparent signs of disease or pathological phenotype. Here, we describe the establishment of our Dnmt1s-transgenic mouse model and propose possibilities for the absence of transgenic protein.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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