Picture from Maduro MF et al. (1996) Gene "Conservation of function and expression of unc-119 from two Caenorhabditis ...."

Fig. 7. X-gal staining of C. elegans hermaphrodites transgenic for unc-ll9::f3gal fusions. Left column, unc-ll9 (ed4); edEx42 (C. briggsae fusion; edEx42 contains the fusion plasmid pDP#MM082 and the rescuing clone pDP#MM016); right column [except (h)], unc-ll9(ed3); edEx43 (C. elegans fusion; edEx43 contains the fusion plasmid pDP#MM081 and pDP#MM016); (h) wild-type animal transgenic for pRF4 (rol-6Dmarker) and pDP#MM082. Despite the nuclear localizing signal (NLS) present in the reporter gene constructs, staining is still visible outside of nuclei (as verified by DAPI staining; data not shown). Panels a and b, comma-stage embryos showing anteriorly localized staining in presumptive neuronal cell precursors; (c) adult and (d) fourth larval stage animals, showing staining in the nerve ring (arrows), pre-anal ganglia (open arrowheads) and ventral nerve cord (filled arrowheads); (e) and (f), adults showing staining in the nerve ring (arrows) and ventral nerve cord (filled arrowheads); (g) and (h), ventral view of hermaphrodite vulva (V) showing the ventral nerve cord and a VC neuron (*). A diffusely staining early embryo (E) is visible in each panel. Methods: Construction of pDP#MM081, predicted to encode the first 101 aa of C. elegans UNC-119, has been described (Maduro and Pilgrim, 1995). For construction of the C. briggsae fusion, two intermediate plasmids were used to generate suit- able restriction sites. The plasmid UR#91 (a gift from W. Wadsworth) contains an EcoRI-HindIII fragment cloned into pBluescript SK+; this fragment was removed from the polylinker of pUC19 which con- tained a 120 bp insertion at the BglII site. UR#91 was digested with EcoRI and BglII (both enzymes BRL) to liberate the 120 bp insertion and part of the pUC polylinker, and allow the insertion of a BamHI- EcoRl fragment from C. briggsae. This intermediate plasmid, pDP#MM070, was digested with HindIII (Pharmacia) and PstI (BRL) to allow its subcloning into similarly digested pPD22.04 (Fire et al., 1990), which is predicted to join the first 130 aa of C. briggsae UNC-119 with bgal.

Picture from Ali Khan L et al. (1997) J Mol Biol "Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ...."

Figure 5. Cellular expression of the klp-3::lacZ fusion gene in transgenic animals. The scale bar represent 20 um. A, A late 'pretzel' stage embryo showing intense staining in the tail of the late stage embryo. However, no staining is seen in early embryos. B, Nuclear staining of DNA using DAPI dye of the same embryo shown in A. C, Intense staining of the pharyngeal marginal cells in the adult head; anterior is to the left. Notice the absence of staining in the second bulb and the pharyngeal intestinal valve cells. The panel also shows a young L1 larva showing very high staining in the head, and the anterior and posterior region of the larval gut. D, DAPI nuclear staining of the animals shown in C. E, A late larva (L3) showing the intense staining in the pharynx, head and in the posterior gut region. Notice that the staining in the gut has been considerably reduced in later larval stages compared to the early L1 larva, shown in C. F, DAPI staining of the same larval animal shown in E. G, Left dorsolateral view of an adult head. Staining of the possible marginal cells in the pharynx is very intense. H, DAPI staining of the same animal shown in G. Methods: microinjection of the klp-3 genomic fragment and the reporter gene: first a 9 kb KpnI/BamHI fragment, which contains about 3 kb of coding region and 6 kb of upstream promoter, was subcloned from the cosmid T09A5 (obtained from the Sanger Centre) into a pBluescript SK(-) vector. From this 9 kb fragment, a 3 kb HindIII-BglII fragment, which contains 1 kb of upstream promotor and 2 kb of coding region was ligated into the HindIII-BamHI site of the lacZ vector pPD16.51 (Fire et al., 1990). The klp-3::lacZ fusion construct (Figure 2C): for microinjection, the klp-3::lacZ fusion gene was injected with a dominant rol-6 cotransformation marker DNA (kindly given by J. Kramer) at a concentration of 75 ug/ml into the syncitial gonad of the N2 hermaphrodites, as described by Tabish et al. (1995). The him-14 ts(it44) mutant allele was used in the mutant rescue experiments with the klp-3 genomic fragment. Injections were performed at the permissive temperature of 15 C, and transformants were tested at the non-permissive temperature of 25 C. The klp-3::lacZ transformants were histochemically stained and analysed essentially as previously described (Tabish et al., 1995).