Download Effects of FGF-4 Growth Factor on Axolotl Fibroblast`s Gene

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.