Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors.
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.
Hozumi
K
K
Department of Immunology, Tokai University School of Medicine, Bohseidai, Isehara, Japan.
Ohtsuka
R
R
Suzuki
D
D
Ando
K
K
Ito
M
M
Nishimura
T
T
Merkenschlager
M
M
Habu
S
S
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*
D018801:CD2 Antigens