Müller cells were isolated, characterized, and maintained in continuous culture for 1 and 5 weeks, yielding phenotypes referred to as
proliferative (GFAP positive, αSMA negative) and
myofibroblastic (GFAP negative, αSMA positive). To evaluate changes in IGFBP biosynthesis associated with phenotype, total RNA harvested from these cultures and normal porcine retina were examined by RT-PCR with IGFBP-specific primers. Normal porcine liver, a source of all six high-affinity IGFBPs, served as a positive control for primer function. Amplimers of the predicted sizes were present in each reaction with liver RNA
(Fig. 1) , confirming successful primer design. Although there was no evidence of IGFBP-1–specific message in normal retina or Müller cells of either phenotype, amplimers for IGFBP-2 through -6 were detected in all three RNA preparations. Northern blot analyses were performed on the same RNA preparation to evaluate transcript size and relative abundance. IGFBP-specific message abundance varied considerably in normal liver, but was consistent with production profiles described for fasting animals
(Fig. 2) .
24 Message levels for the five IGFBP species examined were undetectable or low in retina relative to G3PDH. There was little change between normal retina and
proliferative Müller cells, except for modest increases in message levels for IGFBP-4, -5, and -6. However, the abundance of mRNA for IGFBP-2, -3, -4, and -6 was substantially increased in the
myofibroblastic Müller cell phenotype, whereas expression of IGFBP-5 was reduced. Changes in IGFBP-specific message were confirmed in real-time RT-PCR reactions, using the same primer sets as in
Figure 1 to evaluate, in this case, mRNA abundance between the culture phenotypes. IGFBP-2, -3, -4, and -6 message levels in
myofibroblastic Müller cells increased from 6- to 70-fold compared with the
proliferative phenotype, whereas IGFBP-5 message was reduced by approximately one half
(Table 2) .