Protein transduction as a strategy for evaluating important factors in mammalian sex determination and differentiation.
Protein transduction is a powerful tool to deliver biologically active protein into mammalian cells and whole animals. Transduced proteins are folded properly and can mediate their respective functions in their hosts. To examine the feasibility of applying this strategy to study the molecular events of gonadogenesis, we have studied the kinetics of protein transduction and stability of transduced protein in in vitro mouse gonad culture systems using two reporter proteins, TAT-beta-gal and beta-gal fusion proteins with and without the TAT protein transduction domain (PTD) respectively. Our results indicate that the TAT-PTD was critical and essential for protein transduction to cultured fetal gonads. The TAT-beta-gal reporter entered the cells of the gonads and mesonephros efficiently for both sexes at E11.5 to E15.5 stages examined. The delivered protein persisted in the gonads for an extended period after an initial one-hour transduction. The distribution of the reporter was relatively even in gonads and mesonephros at E11.5 stage for both sexes and at later stages in female. The transduced protein was distributed heterogeneously in male gonads after seminiferous tubule differentiation in which the amount of reporter protein was higher outside than inside the tubules. Nevertheless, we surmise that such protein delivery technique should be useful in studies designed to evaluate the sex determining or differentiating functions of various new protein factors identified by advanced differential screening strategies.
Li
Y
Y
Department of Medicine, VA Medical Center, University of California, San Francisco, San Francisco, CA 94121, USA.
Yue
L
L
Taketo
T
T
Lau
Y-F C
YF
D000818:Animals; D002478:Cells, Cultured; D005260:Female; D006066:Gonads / Q000033:anatomy & histology / Q000196:embryology / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008322:Mammals; D051379:Mice; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012732:Sex Determination Analysis / Q000379:methods*; D012733:Sex Differentiation
D011993:Recombinant Fusion Proteins