Abstract
One of the most thrilling experiments in biology is to introduce a gene of one’s own design into a favorite animal and examine the effect in the transgenic progeny. Methods to construct, transform and monitor transgenes have been available to worm breeders since the pioneering work of Andy Fire and Craig Mello (Fire, 1986; Mello and Fire, 1995) and the introduction of green fluorescent protein (GFP) by Marty Chalfie (Chalfie et al., 1994). Sadly, for many years, the thrill of “seeing green” was denied to worm breeders working on the germline, as transgenes stubbornly refused to express in germ cells. In 1997, Bill Kelly and Andy Fire showed that transgene silencing in the germline is a copy-number driven process (Kelly et al., 1997). Multi-copy transgenes are expressed in the soma but silenced in the germline; in contrast, low-copy transgenes are expressed in both. Today, new transformation methods make it possible to routinely obtain low copy transgenes inserted in the genome. In this chapter, we review these methods and give practical advice for designing and transforming “germline-ready” transgenes.
Highlights
Transgenic solutions for the germline and Fire, 1995) and the introduction of green fluorescent protein (GFP) by Marty Chalfie (Chalfie et al, 1994)
The experiments of Kelly and Fire showed that unlike somatic cells, germ cells are very efficient at silencing genes present in multi-copy
Silencing affects transgene expression in all germ cells, and in the blastomeres of early embryos, which depend primarily on maternal mRNAs and proteins synthesized during oogenesis
Summary
The experiments of Kelly and Fire showed that unlike somatic cells, germ cells are very efficient at silencing genes present in multi-copy. Two other methods yield transgenes that are inserted in the genome, either at random sites [gene-gun transformation (Praitis et al, 2001; Wilm et al, 1999)] or at a pre-selected site [Mos1-mediated Single Copy Insertion (MosSCI) (Frokjaer-Jensen et al, 2008)] These methods yield stable lines that can express in the germline for many generations. Diffusion into the rachis means that transgenic proteins, in particular small reporters like GFP, can end up far from the site of synthesis (Figure 1) For these reasons, when designing germline transgenes, all components (promoter, tag, ORF and 3'UTR) must be considered carefully. When combined with GFP:Histone H2B and tubulin 3'UTR, expression is first detected in the late L1 stage and is maintained in all germ cells through spermatogenesis and oogenesis.
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