title: Construction of a capsule associated protein 10 gene eukaryotic expression vector for RNA interference and confirmation of biologic relevance.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors: Department of Laboratory Medicine, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350005, China. ouqishui@163.com
authors: Qi-shui
authors: QS
authors: Ou
authors: Xiao-ji
authors: XJ
authors: Su
authors: Ni
authors: N
authors: Lin
authors: Ling
authors: L
authors: Jiang
authors: Bin
authors: B
authors: Yang
mesh_terms: D000818:Animals; D002460:Cell Line; D003455:Cryptococcus neoformans / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D010587:Phagocytosis; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: C121601:CAP10 protein, Cryptococcus neoformans; D005656:Fungal Proteins; D034741:RNA, Small Interfering