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1.
CRISPR J ; 5(3): 422-434, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35686982

RESUMO

Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.


Assuntos
Edição de Genes , Zigoto , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Camundongos , Camundongos Knockout , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ribonucleoproteínas/genética , Zigoto/metabolismo
2.
Cancers (Basel) ; 13(21)2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34771750

RESUMO

ERAS is a relatively uncharacterized gene of the Ras superfamily. It is expressed in ES cells and in the first stages of embryonic development; later on, it is silenced in the majority of cell types and tissues. Although there are several reports showing ERAS expression in tumoral cell lines and human tumor samples, it is unknown if ERAS deregulated expression is enough to drive tumor development. In this report, we have generated transgenic mice expressing ERAS in myoepithelial basal cells of the mammary gland and in basal cells of stratified epithelia. In spite of the low level of ERAS expression, these transgenic mice showed phenotypic alterations resembling overgrowth syndromes caused by the activation of the AKT-PI3K pathway. In addition, their mammary glands present developmental and functional disabilities accompanied by morphological and biochemical alterations in the myoepithelial cells. These mice suffer from tumoral transformation in the mammary glands with high incidence. These mammary tumors resemble, both histologically and by the expression of differentiation markers, malignant adenomyoepitheliomas. In sum, our results highlight the importance of ERAS silencing in adult tissues and define a truly oncogenic role for ERAS in mammary gland cells when inappropriately expressed.

3.
Stem Cell Res Ther ; 11(1): 164, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345365

RESUMO

BACKGROUND: CD18 is the common beta subunit of ß2 integrins, which are expressed on hematopoietic cells. ß2 integrins are essential for cell adhesion and leukocyte trafficking. METHODS: Here we have analyzed the expression of CD18 in different subsets of human hematopoietic stem and progenitor cells (HSPCs) from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB) samples. CD34+ cells were classified into CD18high and CD18low/neg, and each of these populations was analyzed for the expression of HSPC markers, as well as for their clonogenity, quiescence state, and repopulating ability in immunodeficient mice. RESULTS: A downregulated membrane expression of CD18 was associated with a primitive hematopoietic stem cells (HSC) phenotype, as well as with a higher content of quiescent cells and multipotent colony-forming cells (CFCs). Although no differences in the short-term repopulating potential of CD18low/neg CD34+ and CD18high CD34+ cells were observed, CD18low/neg CD34+ cells were characterized by an enhanced long-term repopulating ability in NSG mice. CONCLUSIONS: Overall, our results indicate that the downregulated membrane expression of CD18 characterizes a primitive population of human hematopoietic repopulating cells.


Assuntos
Células da Medula Óssea , Células-Tronco Hematopoéticas , Animais , Antígenos CD34/genética , Medula Óssea , Sangue Fetal , Humanos , Camundongos
4.
Int J Oral Sci ; 12(1): 1, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31900382

RESUMO

Odontogenic tumours are a heterogeneous group of lesions that develop in the oral cavity region and are characterized by the formation of tumoural structures that differentiate as teeth. Due to the diversity of their histopathological characteristics and clinical behaviour, the classification of these tumours is still under debate. Alterations in morphogenesis pathways such as the Hedgehog, MAPK and WNT/ß-catenin pathways are implicated in the formation of odontogenic lesions, but the molecular bases of many of these lesions are still unknown. In this study, we used genetically modified mice to study the role of IKKß (a fundamental regulator of NF-κB activity and many other proteins) in oral epithelial cells and odontogenic tissues. Transgenic mice overexpressing IKKß in oral epithelial cells show a significant increase in immune cells in both the oral epithelia and oral submucosa. They also show changes in the expression of several proteins and miRNAs that are important for cancer development. Interestingly, we found that overactivity of IKKß in oral epithelia and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins (p53, or p16 and p19), leads to the appearance of odontogenic tumours that can be classified as ameloblastic odontomas, sometimes accompanied by foci of secondary ameloblastic carcinomas. These tumours show NF-κB activation and increased ß-catenin activity. These findings may help to elucidate the molecular determinants of odontogenic tumourigenesis and the role of IKKß in the homoeostasis and tumoural transformation of oral and odontogenic epithelia.


Assuntos
Células Epiteliais/metabolismo , Genes Supressores de Tumor , Quinase I-kappa B/biossíntese , Mucosa Bucal/patologia , Tumores Odontogênicos/patologia , Odontoma/patologia , RNA Mensageiro/genética , Animais , Western Blotting , Células Epiteliais/patologia , Citometria de Fluxo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Transgênicos , Mucosa Bucal/metabolismo , Tumores Odontogênicos/metabolismo , Odontoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
5.
Hum Gene Ther ; 27(9): 668-78, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27056660

RESUMO

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene and is characterized by recurrent and life-threatening bacterial infections. These mutations lead to defective or absent expression of ß2 integrins on the leukocyte surface, compromising adhesion and extravasation at sites of infection. Three different lentiviral vectors (LVs) conferring ubiquitous or preferential expression of CD18 in myeloid cells were constructed and tested in human and mouse LAD-I cells. All three hCD18-LVs restored CD18 and CD11a membrane expression in LAD-I patient-derived lymphoblastoid cells. Corrected cells recovered the ability to aggregate and bind to sICAM-1 after stimulation. All vectors induced stable hCD18 expression in hematopoietic cells from mice with a hypomorphic Itgb2 mutation (CD18(HYP)), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18(HYP) recipients. hCD18(+) hematopoietic cells from transplanted CD18(HYP) mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18(HYP) mice subjected to two different inflammation models demonstrated that the LV-mediated gene therapy completely restored neutrophil extravasation in response to inflammatory stimuli. Finally, these vectors were able to correct the phenotype of human myeloid cells derived from CD34(+) progenitors defective in ITGB2 expression. These results support for the first time the use of hCD18-LVs for the treatment of LAD-I patients in clinical trials.


Assuntos
Antígenos CD18/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Lentivirus/genética , Síndrome da Aderência Leucocítica Deficitária/terapia , Animais , Antígenos CD34/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Síndrome da Aderência Leucocítica Deficitária/genética , Camundongos , Neutrófilos/citologia , Neutrófilos/metabolismo
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