Publicação
Development of a new and rapid method to introduce heterologous DNA sequences into the Plasmodium genome
| Resumo: | Malaria is one of the most important parasitic diseases nowadays with a reported 243 million cases each year and almost half of the world’s population at risk of contracting the disease. The development of new and more effective drugs as well as a prophylactic vaccine is imperative and even though, in the recent years, several advances have been made with some vaccine candidates there is still no vaccine available. Genome manipulation and reverse genetics constitute powerful tools that contributed to these advances. Together, these techniques allow a better understanding of parasite-host interactions as well as new insight into gene function and expression. With the help of cloning and transfection technologies we aim to develop the proposed Swift Knock-in Markerfree (SKIM) method, a more efficient way to knock-in and/or knock-out genes of interest that overcomes some of the limitations of previously described transfection technologies namely, the time needed to obtain transfectants, low transfection efficiencies and the recycling of the drug resistance markers. The technique is based on a motherline expressing a fused positive/negative selectable marker and, through homologous recombination by double crossover, a heterologous DNA sequence replaces the selectable marker cassette and parasites can be selected by negative selection. We tested the viability of the technique and optimized the negative selection of the parasites with a fluorescent construct – our results show that not only through the SKIM method successful integration is achieved but also the transfection efficiency is high – 93% efficiency with 4 consecutive days of negative selection. Also, we use the technique to express transmission-blocking antigens – P. falciparum and P. berghei P25 and P48/45 – in parallel to the traditional positive selection transfection, with which we were able to introduce most of the antigens. |
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| Autores principais: | Pissarra, Joana da Silva, 1987- |
| Assunto: | Biologia molecular Malária Plasmodium Parasitas Teses de mestrado - 2010 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Malaria is one of the most important parasitic diseases nowadays with a reported 243 million cases each year and almost half of the world’s population at risk of contracting the disease. The development of new and more effective drugs as well as a prophylactic vaccine is imperative and even though, in the recent years, several advances have been made with some vaccine candidates there is still no vaccine available. Genome manipulation and reverse genetics constitute powerful tools that contributed to these advances. Together, these techniques allow a better understanding of parasite-host interactions as well as new insight into gene function and expression. With the help of cloning and transfection technologies we aim to develop the proposed Swift Knock-in Markerfree (SKIM) method, a more efficient way to knock-in and/or knock-out genes of interest that overcomes some of the limitations of previously described transfection technologies namely, the time needed to obtain transfectants, low transfection efficiencies and the recycling of the drug resistance markers. The technique is based on a motherline expressing a fused positive/negative selectable marker and, through homologous recombination by double crossover, a heterologous DNA sequence replaces the selectable marker cassette and parasites can be selected by negative selection. We tested the viability of the technique and optimized the negative selection of the parasites with a fluorescent construct – our results show that not only through the SKIM method successful integration is achieved but also the transfection efficiency is high – 93% efficiency with 4 consecutive days of negative selection. Also, we use the technique to express transmission-blocking antigens – P. falciparum and P. berghei P25 and P48/45 – in parallel to the traditional positive selection transfection, with which we were able to introduce most of the antigens. |
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