The twisted cuticle of roller animals can also complicate live analysis of some cell types. In addition, rollers (especially males) have reduced mating efficiency. The roller phenotype is suppressed in some "dumpy" ( dpy) and "uncoordinated" ( unc) mutant backgrounds. This roller phenotype is very easy to spot in a simple dissecting microscope, although there are a few potential disadvantages. This plasmid encodes a mutant collagen ( rol-6( su1006)) that induces a dominant "roller" phenotype, where animals corkscrew around in circles ( Kramer et al., 1990 Mello et al., 1991). For microinjection techniques, the popular choice is the pRF4 plasmid (Protocols 2 and 3). Some commonly used marker genes are listed in Table 1. For some mutant rescue experiments, a co-injected marker may not be necessary but is usually advisable as a positive control for transformation. Other selectable markers rescue lethal or non-lethal mutations and require use of specific mutant strains as transformation hosts. Transformation markers that induce a dominant phenotype allow transformation of any worm strain as long as the host's phenotype does not interfere with the marker-induced phenotype. The protocols below generally come from a single lab, although a few alternative modifications are included.ĭNA transformation techniques typically require co-transformation with a scoreable or selectable marker gene. In addition, different labs often have distinct preferences and unique modifications of these methods. For an excellent and more thorough analysis of these issues see Mello and Fire (1995). This chapter focuses on the methods themselves and on some practical considerations but does not include extensive discussions of transgene dynamics, vectors, or theory. Protocols for integration of extrachromosomal arrays into the genome are also included. elegans, and for generating transgenic nematode strains by both microinjection and gene bombardment. This chapter contains protocols for microinjection of C. For transformation, the relatively new technique of gene bombardment is fast becoming a popular alternative because transgenes are often integrated into the genome at low copy number, which offers a number of advantages as described below ( Praitis et al., 2001 Berezikov et al., 2004). Moreover, microinjection is a very effective approach to RNA interference (see Reverse genetics), and can be used to deliver synthetic mRNAs or other molecules directly to cells ( Kimble et al., 1982 Bossinger and Schierenberg, 1992 Evans et al., 1994). Microinjection is a proven and relatively simple method for introducing DNA into worms ( Mello et al., 1991 Mello and Fire, 1995). However, homologous recombination into endogenous loci can occur at low frequency suggesting that targeted gene replacement is possible ( Broverman et al., 1993 Berezikov et al., 2004). Current transformation techniques generate large extrachromosomal DNA arrays, or cause "random" integration of transgenes into the genome. Furthermore, transgenes can be powerful tools for the design of new genetic screens. Transformation is used to clone genes by mutant rescue, to over-express or ectopically express genes, to express tagged proteins, to study structure/function of protein domains, and to analyze DNA or RNA regulatory elements. DNA transformation and microinjection are essential tools for C.
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