Meeting Abstract

P2.91  Jan. 5  Development and Use of Real-time PCR for Examining Sox9 Expression in the Red-eared Slider Turtle DODD, K.L.**; WIBBELS, T.; Univ. of Alabama, Birmingham; Univ. of Alabama, Birmingham kdodd@uab.edu

All crocodilians, most turtles, some snakes, and the tuatara exhibit temperature-dependent sex determination (TSD) in which the incubation temperature of the egg determines the sex of the embryo. The molecular physiology involved in sex determination and sex differentiation of reptiles with TSD is currently being explored. One gene that may play a role is Sox9. Sox9 is believed to be involved in testicular differentiation in mammals. Further, temperature has been shown to regulate Sox9 in the gonads of a turtle with TSD, Lepidochelys olivacea, during in vitro studies. During the current study, SOX9 expression was examined during sex determination and sex differentiation in the red-eared slider, Trachemys scripta, a turtle with TSD. A real-time PCR was developed to evaluate the expression of SOX9 in this turtle. First, a portion of Sox9 was cloned, and a 100 bp fragment of a highly conserved area was selected to create a standard curve ranging from 50 to 0.00005ng. A reverse-transcriptase reaction was preformed on sample total RNA. Then, using real-time PCR, the same 100 bp fragment was amplified from the total cDNA. Quantities of the 100 bp fragment in the samples are compared to the standard curve in order to determine the relative quantities of SOX9 in the samples. Samples were taken from embryonic stages 17, 19, 21, 23, and 26. Results showed no significant difference in expression levels during the thermo-sensitive period. However, an increase in Sox9 expression occurs soon after the thermo-sensitive period, coinciding with the time when morphological differentiation of the testis is apparent. Thus, the results suggest that Sox9 may play a role in testis differentiation rather than sex determination in the red-eared slider.