Construction of a capsule associated protein 10 gene eukaryotic expression vector for RNA interference and confirmation of biologic relevance / 中华医学杂志(英文版)
Chinese Medical Journal
;
(24): 2741-2745, 2011.
Artículo
en Inglés
| WPRIM
| ID: wpr-292813
ABSTRACT
<p><b>BACKGROUND</b>The capsule associated protein 10 gene (cap10) is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus (C.) neoformans. In this study, we aimed to construct an short hairpin RNA (shRNA) expression vector targeting C. neoformans cap10 gene expression and confirm its biologic relevance.</p><p><b>METHODS</b>A pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method.</p><p><b>RESULTS</b>The expression of cap10 in C. neoformans cells transfected with ps4.1 neo-cap10 ((175,535.00 ± 47,004.00) copies/µl) was lower than that of cells transfected with the empty vector ((512,698.89 ± 32,318.02) copies/µl) and mock transfected cells ((562,931.66 ± 65,928.41) copies/µl). The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-cap10 (0.21 ± 0.02, (19.06 ± 1.66)%) than for the control experimental group (0.08 ± 0.02, (6.57 ± 1.23)%) and the blank experimental group ((0.07 ± 0.01), (5.89 ± 1.07)%) (P < 0.05).</p><p><b>CONCLUSIONS</b>The cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans cap10 gene is effective, and it can induce phagocytosis of C. neoformans.</p>
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Fagocitosis
/
Plásmidos
/
Proteínas Fúngicas
/
Transfección
/
Línea Celular
/
Reacción en Cadena de la Polimerasa
/
Cryptococcus neoformans
/
ARN Interferente Pequeño
/
Interferencia de ARN
/
Vectores Genéticos
Límite:
Animales
Idioma:
Inglés
Revista:
Chinese Medical Journal
Año:
2011
Tipo del documento:
Artículo
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