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Download Effects of FGF-4 Growth Factor on Axolotl Fibroblast`s Gene
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21126 Effects of FGF-4 Growth Factor on Axolotl Fibroblast Gene Expression Author: Banafsheh H. Noosha Mentor: Dr. David Gardiner Although vertebrae develop limbs as embryos, only salamanders (urodele amphibians) are able to regenerate limbs as adults. Recent studies of salamanders indicate how gene expression varies amongst different stages of regeneration in vivo; however, little is known about regulating gene expression in vitro. Such information is important in designing strategies to induce the expression of regeneration genes in animals that normally do not regenerate, such as humans. Growth factors alter gene expression, and thus it is important to study how different growth factors and their varying concentrations affect the genes involved in limb regeneration. Fibroblast cells from axolotl tissue were grown in culture medium (60%L-15 + 5%FBS + 1%ITS). Cells were plated on T75 tissue culture flasks and incubated for two weeks as controls. Following this two-week period of growth (cell division), cells were supplied with new culture medium. Half of the cells were treated with 100 ng/mL of FGF-4 growth factor and half were treated in culture medium without exogenous FGF-4. After 18-24 hours, cells were trypsinzed, centrifuged, and lysed to extract total RNA (Qiagen’s RNeasy MiniPrep kit). Total RNA was quantified, Reverse Transcribed, and run in a Polymerase Chain Reaction, in which primers were designed based on the genes to be studied. These genes were GAPDH, a normalizing control, TGF-1 and MMP-9, which have been implicated as being involved in regeneration. Agarose gel electrophoresis revealed the amount of amplified experimental cDNA relative to the amount of GAPDH. Preliminary results indicate that MMP-9 is expressed at high levels in control cells, and that TGF-1 is expressed at much lower levels. More specific responses are currently being analyzed, which are critical in identifying key regulatory molecules for vertebrate limb regeneration.