Chan Robin F, Lewellyn Lara, DeLoyht Jacqueline M, Sennett Kristyn, Coffman Scarlett, Hewitt Matthew, Bettinger Jill C, Warrick John M, Grotewiel Mike
Molecular Biology and Genetics Program, Virginia Commonwealth University, Richmond, Virginia; Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia.
Alcohol Clin Exp Res. 2014 Jun;38(6):1582-93. doi: 10.1111/acer.12421. Epub 2014 May 30.
The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol (EtOH)-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white(mini-w), a derivative of the endogenous gene white(w). Whether the mini-w transgenic marker or the endogenous w gene influences behavioral responses to acute EtOH exposure in flies has not been systematically investigated.
We manipulated mini-w and w expression via (i) transposons marked with mini-w, (ii) RNAi against mini-w and w, and (iii) a null allele of w. We assessed EtOH sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of EtOH) and an assay based on EtOH-induced sedation.
In eRING assays, EtOH-induced impairment of climbing correlated inversely with expression of the mini-w marker from a series of transposon insertions. Additionally, flies harboring a null allele of w or flies with RNAi-mediated knockdown of mini-w were significantly more sensitive to EtOH in eRING assays than controls expressing endogenous w or the mini-w marker. In contrast, EtOH sensitivity and rapid tolerance measured in the EtOH sedation assay were not affected by decreased expression of mini-w or endogenous w in flies.
EtOH sensitivity measured in the eRING assay is noticeably influenced by w and mini-w, making eRING problematic for studies on EtOH-related behavior in Drosophila using transgenes marked with mini-w. In contrast, the EtOH sensitivity assay described here is a suitable behavioral paradigm for studies on EtOH sensitivity and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-w.
果蝇已被广泛用于研究与乙醇（EtOH）相关行为的遗传机制。过去许多关于果蝇的研究，包括我们实验室的研究，都使用携带遗传表型标记mini-white（mini-w）的转座子来操纵基因表达，mini-white是内源基因white（w）的衍生物。mini-w转基因标记或内源w基因是否会影响果蝇对急性EtOH暴露的行为反应尚未得到系统研究。
我们通过以下方式操纵mini-w和w的表达：（i）用mini-w标记的转座子；（ii）针对mini-w和w的RNA干扰；（iii）w的无效等位基因。我们使用先前描述的eRING试验（基于在EtOH存在下的攀爬）和基于EtOH诱导镇静的试验来评估EtOH敏感性和耐受性。
在eRING试验中，EtOH诱导的攀爬损伤与一系列转座子插入的mini-w标记的表达呈负相关。此外，携带w无效等位基因的果蝇或mini-w被RNA干扰介导敲低的果蝇在eRING试验中对EtOH的敏感性明显高于表达内源w或mini-w标记的对照果蝇。相比之下，在EtOH镇静试验中测量的EtOH敏感性和快速耐受性不受果蝇中mini-w或内源w表达降低的影响。
在eRING试验中测量的EtOH敏感性明显受w和mini-w的影响，这使得eRING对于使用标记有mini-w的转基因研究果蝇中与EtOH相关行为存在问题。相比之下，这里描述的EtOH敏感性试验是研究果蝇EtOH敏感性和快速耐受性的合适行为范式，包括那些使用广泛可用的标记有mini-w的转基因的研究。
