title: Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors.
abstract: To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.
authors.affiliation: Department of Immunology, Tokai University School of Medicine, Bohseidai, Isehara, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Hozumi
authors.forename: R
authors.initials: R
authors.lastname: Ohtsuka
authors.forename: D
authors.initials: D
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Ando
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: T
authors.initials: T
authors.lastname: Nishimura
authors.forename: M
authors.initials: M
authors.lastname: Merkenschlager
authors.forename: S
authors.initials: S
authors.lastname: Habu
mesh_terms: D000818:Animals; D018801:CD2 Antigens / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D005822:Genetic Vectors; D051379:Mice; D009924:Organ Culture Techniques; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology*; D013950:Thymus Gland; D014162:Transfection / Q000379:methods*
chemical_list: D018801:CD2 Antigens