Department of Genetics, Evolution & Environment, University College London, London, UK.
BMC Mol Biol. 2010 Nov 16;11:86. doi: 10.1186/1471-2199-11-86.
Stalk-eyed flies of the family Diopsidae have proven to be an excellent model organism for studying the evolution of ornamental sexual traits. In diopsid flies the eyes and antennae are borne at the end of lateral head projections called 'eye-stalks'. Eyespan, the distance between the eyes, and the degree of sexual dimorphism in eyespan vary considerably between species and several sexually dimorphic species show sexual selection through female mate preference for males with exaggerated eyespan. Relatively little is known about the molecular genetic basis of intra- or inter-species variation in eyespan, eye-stalk development or growth regulation in diopsids. Molecular approaches including comparative developmental analyses, EST screening and QTL mapping have identified potential candidate loci for eyespan regulation in the model species Teleopsis dalmanni. Functional analyses of these genes to confirm and fully characterise their roles in eye-stalk growth require the development of techniques such as germline transformation to manipulate gene activity in vivo.
We used in vivo excision assays to identify transposon vector systems with the activity required to mediate transgenesis in T. dalmanni. Mariner based vectors showed no detectable excision while both Minos and piggyBac were active in stalk-eyed fly embryos. Germline transformation with an overall efficiency of 4% was achieved using a Minos based vector and the 3xP3-EGFP marker construct. Chromosomal insertion of constructs was confirmed by Southern blot analysis. Both autosomal and X-linked inserts were recovered. A homozygous stock, established from one of the X-linked inserts, has maintained stable expression for eight generations.
We have performed stable germline transformation of a stalk-eyed fly, T. dalmanni. This is the first transgenic protocol to be developed in an insect species that exhibits an exaggerated male sexual trait. Transgenesis will enable the development of a range of techniques for analysing gene function in this species and so provide insight into the mechanisms underlying the development of a morphological trait subject to sexual selection. Our X-linked insertion line will permit the sex of live larvae to be determined. This will greatly facilitate the identification of genes which are differentially expressed during eye-stalk development in males and females.
柄眼蝇科的柄眼蝇已被证明是研究装饰性性特征进化的极好模式生物。在柄眼蝇中，眼睛和触角位于称为“眼柄”的头部侧突末端。眼距，即眼睛之间的距离，以及眼距的性二型性在物种间差异很大，几个性二型物种表现出通过雌性对具有夸张眼距的雄性的配偶偏好进行性选择。关于柄眼蝇中眼距、眼柄发育或生长调节的种内或种间变异的分子遗传基础相对知之甚少。包括比较发育分析、EST 筛选和 QTL 图谱的分子方法已经确定了模型物种 Teleopsis dalmanni 中眼距调节的潜在候选基因座。这些基因的功能分析，以确认并充分描述它们在眼柄生长中的作用，需要开发技术，如生殖系转化，以在体内操纵基因活性。
我们使用体内切除测定来鉴定具有在 T. dalmanni 中介导转基因所需活性的转座子载体系统。基于 Mariner 的载体没有检测到切除，而 Minos 和 piggyBac 在柄眼蝇胚胎中都具有活性。使用基于 Minos 的载体和 3xP3-EGFP 标记构建体实现了 4%的整体生殖系转化效率。通过 Southern blot 分析证实了构建体的染色体插入。回收了常染色体和 X 连锁插入物。从一个 X 连锁插入物中建立的纯合株系已经维持了 8 代的稳定表达。
我们已经对柄眼蝇 T. dalmanni 进行了稳定的生殖系转化。这是在表现出夸张雄性性特征的昆虫物种中开发的第一个转基因方案。转基因将使一系列分析该物种中基因功能的技术得以发展，从而深入了解受性选择影响的形态特征的发展机制。我们的 X 连锁插入系将允许确定活幼虫的性别。这将极大地促进鉴定在雄性和雌性眼柄发育过程中差异表达的基因。
