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1.
PLoS One ; 9(3): e91892, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24618785

RESUMO

The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen-thawed spermatozoa, producing live pups. The ability to cryopreserve mouse gametes for LAIVF may facilitate management of large-scale transgenic mouse production facilities.


Assuntos
Fertilização in vitro , Fertilização , Oócitos/citologia , Espermatozoides/citologia , Animais , Movimento Celular , Sobrevivência Celular , Criopreservação , Embrião de Mamíferos , Feminino , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Taxa de Gravidez
2.
J Mol Biol ; 422(4): 532-44, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22706026

RESUMO

Dysregulation of epidermal growth factor receptor (EGFR) is a hallmark of many epithelial cancers, rendering this receptor an attractive target for cancer therapy. Much effort has been focused on the development of EGFR-directed antibody-based therapeutics, culminating in the clinical approval of the drugs cetuximab and panitumumab. Unfortunately, the clinical efficacy of these drugs has been disappointingly low, and a particular challenge to targeting EGFR with antibody therapeutics has been resistance, resulting from mutations in the downstream raf and ras effector proteins. Recent work demonstrating antibody cocktail-induced synergistic downregulation of EGFR motivated our design of cetuximab-based antibody-fibronectin domain fusion proteins that exploit downregulation-based EGFR inhibition by simultaneously targeting multiple receptor epitopes. We establish that, among our engineered multiepitopic formats, trans-triepitopic antibody fusions demonstrate optimal efficacy, inducing rapid EGFR clustering and internalization and consequently ablating downstream signaling. The combined effects of EGFR downregulation, ligand competition, and immune effector function conspire to inhibit tumor growth in xenograft models of cetuximab-resistant BRAF and KRAS mutant cancers. Our designed triepitopic constructs have the potential to enhance the efficacy and expand the scope of EGFR-directed therapies, and our multiepitopic may be readily applied to other receptor targets to formulate a new class of antibody-based therapeutics.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Cetuximab , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Feminino , Fibronectinas/imunologia , Fibronectinas/farmacologia , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Camundongos , Mutação , Neoplasias/genética , Neoplasias/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Proteínas ras/metabolismo
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