Heasman et al. (2000); Nutt et al. (2001)
A. Injection Sample Preparation
Many useful reagents are available commercially from sources such as Molecular Probes or Cytoskeleton, Inc., and can be injected directly into blastomeres. For example, fluorescent tracers, calcium indicators, and proteins are used to mark and measure dynamic changes in live cells. Because the volume injected into blastomeres is typically a small fraction of the total, most dextrans can be diluted in DEPC water or phosphate-buffered saline (PBS) prior to injection. Glycerol-free proteins should also be centrifuged at 100,000 x g for 20min to remove precipitates in the solution using a Beckman airfuge or equivalent. We have found that most fluorescent tracers and indicators do not alter normal development if injected at reasonable levels into blastomeres (Table 1), including, FITC-dextran, caged-FITC dextran, TRITC-dextran, and Oregon green BAPTA dextran. Fluorescent tracers are often used to examine cell motility, axon outgrowth, and calcium signaling in live cells (Collazo et al., 1993; Davidson and Keller, 1999; Gomez and Spitzer, 1999; Wallingford et al., 2001) or to identify cells after fixation using lysine-fixable forms of fluorescent dextrans (Lin and Szaro, 1995; Moran-Rivard et al., 2001). Fluorescent fusion proteins are also injected readily into blastomeres and combine with endogenous proteins to allow examination of dynamic protein distribution in living cells (Tanaka and Kirschner, 1995; Noguchi and Mabuchi, 2001). However, with the cloning of the green and red fluorescent proteins (GFP and RFP, respectively (Prasher et al., 1992; Matz et al., 1998), and the molecular engineering of several spectral variants (Heim and Tsien, 1996), new possibilities involving molecular expression rather than direct protein injection exist.
The injection of DNA or RNA encoding for fluorescent (GFP, RFP, etc.), functional, or fluorescence resonance energy transfer (FRET)-based reporter proteins is one of the most useful, versatile, and cost-effective ways to label, modify, or make physiological measurements in developing Xenopus neurons. A particularly effective plasmid for driving expression in Xenopus is the pCS2 plasmid. The pCS2 expression vector, originally designed by Dave Turner and Ralph Rupp, was based on the backbone of pBluescript II KS+. This vector combines the simian CMV IE94 promoter with the SV40 polyadenylation site and two polylinker regions. An SP6 promoter in the 5-untranslated region of the CMV promoter allows for the in vitro RNA synthesis of sense sequences inserted into the first polylinker. The second polylinker provides several choices to linearize the vector for the transcription of sense RNA and for subcloning in different transcripts. A T7 promoter site outside the second polylinker allows for the transcription of antisense RNA sequences. RNA transcribed from the SP6 promoter through the SV40 poly(A)site produces highly stable mRNA that is translated efficiently when injected into Xenopus embryos. Alternatively, expression from injected pCS2 DNA is also possible, but results in delayed and highly mosaic expression (see later). Numerous additions to the basic CS2 vector exist, including variants expressing myc, ^-galactosidase, and fluorescent protein tags.
Plasmid DNA and mRNA each provide unique opportunities to ectopically express proteins in developing embryos. Dilute solutions of mini-prep DNA in DEPC water (~100pg/nl) is normally pure enough to inject; however, less than 100 pg per blastomere should be introduced, as DNA can be toxic above this level. 5-capped mRNA is transcribed in vitro from linearized CS2 DNA using the SP6 mMessage machine kit (Ambion) and is diluted in DEPC water (~1 ng/nl). Up to 5 ng per blastomere mRNA can be tolerated; however, the level of expression is often so great that reducing the amount of injected RNA is necessary. For example, to express GFP fusion proteins, we typically inject just 100-300 pgmRNA. Because early Xenopus embryos express proteins exclusively from maternally derived mRNA, injected DNA does not begin to be expressed until the early gastrula stages at around 9 h postfertilization (hpf). Therefore, DNA injections result in a highly mosaic expression that is delayed as compared to RNA (Fig. 1). This delayed and limited expression can be useful if the expressed protein has deleterious effects on early embryos. However, even by 24 hpf, the level of protein expression from DNA injections is less than that after RNA injections. Contrary to DNA, RNA begins being expressed within 1 h of injection and is expressed homogeneously at high levels in all cells derived from the injected blastomere.
Embryos can be injected with plasmid DNA or mRNA at the single cell stage and beyond. Early stage injections lead to a greater proportion of labeled cells but at the cost of increased developmental defects and embryo death. Injections at the 1 to 4 cell stage are favorable for in vitro and cell transplantation experiments where large numbers of expressing cells of varying types are desired. However, embryo injections at the 32 and 64 cell stage are well suited for the labeling of particular classes of neurons based on established lineage maps (Jacobson and Hirose, 1981; Moody, 1989). Specific regions of the spinal cord and brain can be targeted, leaving individual classes of neurons in these regions identifiable in vivo. The dorsoventral pigmentation patterns of blastomeres are used to identify the precursors of particular cell types.
On the day of injections, fresh eggs obtained from ovulating female frogs are fertilized in vitro using isolated testes taken from sexually mature male frogs. Detailed methods of how to obtain and fertilize Xenopus eggs have been described elsewhere (Sive et al., 2000). This section describes the injection of blastula stage embryos between the 2 and the 64 cell stages. The initial cell divisions proceed rapidly over the first 3.5/hpf at room temperature, but can be slowed slightly by cooling embryos between 16 and 22° C. However, because healthy ovulating females often lay eggs throughout the day, we prefer to fertilize multiple batches of eggs staggered by 1-2 h.
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