RETROVIRAL GENE-TRANSFER IN CHONDROGENIC LIMB BUD MICROMASS CULTURES

We report development of a model of retroviral gene transduction in hi gh-density limb bud cell micromass culture. The replication competent avian retrovirus RCAS BP (A) carrying the human placental alkaline pho sphatase gene (RCAs AP) was used as a marker for retroviral infection and spread. The final protocol balances the need to allow time for ret roviral integration and gene transduction against loss of chondrogenic potential when limb bud cells are plated at low density. It includes: (i) incubation of the dissociated limb bud cells with RCAS virus for 2 h followed by low-density culture for 48 h to allow retroviral gene expression; and (ii) secondary replating as high-density micromass cul ture to initiate chondrogenesis. The pattern and level of chondrogenes is in the retrovirus-transduced micromass cultures is similar to regul ar micromass cultures. At least 40%-50% of cells express the retrovira l-transduced genes 24 h after high-density plating. This new approach facilitates ectopic gene expression in micromass culture, enabling mol ecular dissection of chondrogenesis and serves as a model for gene tra nsduction in other organotypic culture.