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Cloning and Expression of Melanocortin 1 Receptor (Mc1r)

Melanoma is a malignant tumour that originates mainly from melanocytes in the skin and mucous membranes . Nowadays, one of the most reliable markers for future treatment of this disease is the Melanocortin 1 Receptor (MC1R), a G-coupled receptor involved in several pathways leading to develop and progression of Melanoma. The main aim of this study is to isolate, clone and express MC1R gene using an innovative technology denominated “GATEWAY® TECHNOLOGY” (Invitrogen™). This is to create the possibility of moving DNA sequences and making them suitable for protein expression and functional analysis. Firstly we have isolated and amplified the MC1R coding region (954bp, intronless gene, NCBI Entrez Gene) by Polymerase Chain reaction (PCR). The isolated gene has been cloned in an entry vector by pENTR™ Directional TOPO® Cloning. The final step was moving MC1R into the destination vector pDEST™27 (Invitrogen™) by “LR Recombination” to generate the expression clone. Pdest ™27 is a Gateway®-adapted destination vector useful for cloning and high-level expression of native or tagged recombinant proteins in mammalian cells. Further studies can focus on MC1R protein expression, functional analysis, investigation and identification of the mechanisms that regulate the expression of MC1R and its roles in the etiology and treatment of Melanoma.

Mostra/Nascondi contenuto.
SUMMARY Melanoma is a malignant tumor that originates mainly from melanocytes in the skin and mucous membranes . Nowadays, one of the most reliable markers for future treatment of this disease is the Melanocortin 1 Receptor (MC1R), a G-coupled receptor involved in several pathways leading to develop and progression of Melanoma. The main aim of this study is to isolate, clone and express MC1R gene using an innovative technology denominated “GATEWAY® TECHNOLOGY” (Invitrogen™). This is to create the possibility of moving DNA sequences and making them suitable for protein expression and functional analysis. Firstly we have isolated and amplified the MC1R coding region (954bp, intronless gene, NCBI Entrez Gene) by Polymerase Chain reaction (PCR). The isolated gene has been cloned in an entry vector by pENTR™ Directional TOPO® Cloning. The final step was moving MC1R into the destination vector pDEST™27 (Invitrogen™) by “LR Recombination”. pDEST™27 is a Gateway®-adapted destination vector useful for cloning and high-level expression of native or tagged recombinant proteins in mammalian cells. Further studies can focus on MC1R protein expression, functional analysis, investigation and identification of the mechanisms that regulate the expression of MC1R and its roles in the etiology and treatment of Melanoma.

Tesi di Specializzazione/Perfezionamento

Autore: Andrea Nervegna Contatta »

Composta da 61 pagine.

 

Questa tesi ha raggiunto 240 click dal 01/09/2009.

Disponibile in PDF, la consultazione è esclusivamente in formato digitale.