Retroviral gene transduction in limb bud micromass cultures.

1. Introduction Since Solursh and his coworkers set up the limb bud micromass cultures in 1977 (1), this procedure has become a major model to analyze cellular and molecular event involved in chondrogenesis (2–7). Recently, RCAS retroviral vectors have been developed that can infect chicken embryos in ovo. These have advanced our understanding in limb bud patterning remarkably (8). Here we describe a newly developed protocol to merge these two procedures, which will make micromass culture an even more powerful model for the analysis of chondrogenic mechanisms. Micromass culture has been used to study the role of growth factors, enzyme modu-lators, drugs, and adhesion molecules in chondrogenesis (3–7). These studies have been facilitated by the development of serum-free media (9). However, because some sig-naling molecules are intracellular, it has been difficult to dissect the chondrogenic pathways at a molecular level. To manipulate gene expression in micromass cultures, we have used electroporation to introduce exogenous genes into limb bud cells (7). However, this leads to a high mortality rate of cells, so we have been continuing to search for better procedures. With the advent of gene therapy (10), retroviral mediated gene delivery has become a more mature technology. It has several advantages. The initial infection is not toxic to the cell, the gene expression is more stable than other procedures, and the percentage of infected cells will increase over time if the virus is replication competent. It takes approx 18 h after retrovirus infection for the gene to be expressed. Because precarti-lage condensation formation in micromass cultures starts within 3 h of plating, we would like to have most, if not all, cells expressing the transgene at this stage. To achieve this goal, we have devised a novel two-stage micromass culture, with a low-density plating window allowing retroviral gene transduction into primary limb bud cells, and a regular high-density plating allowing cells to differentiate (see Note 1). This strategy was used successfully to demonstrate the dual action of SHH in chondrogenesis (11; see Note 2) and the arrest effect of Wnt 7a at early stages of precartilage condensation formation (16).