LacZ expression

Kress, C., Vogels, R DeGraff, W Bonnerot, C., Meijlink, F and Deschamps, J. (1990). Hox 2.3 upstream sequences mediate lacZ expression in intermediate mesoderm derivatives of transgenic mice. Development 109 775-786. 

With the optimized lipid composition (opDC ePC DOPE eSph Choi DC-Chol = 5 5 5 12 3), the HVJ-cationic liposomes showed 100 to 800 times greater transfection efficiency in vitro compared with the conventional HVJ-PS liposomes. The presence of serum (10% FCS) in the transfection mixture did not decrease luciferase activity significantly. Even 70% FCS reduced the activity by less than 40%. LacZ gene expression showed that transfection efficiency of BHK-21 cells by optimized HVJ-cationic liposomes (opDC) and by conventional HVJ-cationic liposomes (DC) was 90-100% and 50-60%, respectively. With conventional HVJ-anionic liposomes (PS), LacZ expression was found in only 1-3% of the cells. The optimized HVJ-cationic liposomes were also much more effective for the transfer of FITC-labeled ODNs to cultured cells [16]. 

Tissue-cultured ceUs or animal tissues are prepared, fixed, and stained for lacZ expression as described in preceding sections. Instead of storing the cells in 0.02% sodium azide in PBS, the cells are rinsed again with PBS. ALP staining should be performed as described in the previous section, starting with the 65°C heat treatment and reducing the number of PBS washes that follow from three to one (Lin et al., 1992). 

The Arabidopsis AtAux2-Il promoter-LacZ reporter gene is expressed in elongating regions of roots, etiolated hypocotyls, and anther filaments of transgenic Arabidopsis plants [156]. LacZ expression is also detected in the root cap and at sites of lateral root... 

As in the commercial PN strains, glucose and sucrose were found to repress PN production in A. nidulans. Compared to lactose-grown cultures, ipnA lacZ expression was reduced with these two carbon sources. " The glucose-dependent repression of many genes in primary metabolism is mediated by the well-characterized carbon catabolite repression (ere) system. While CreA mediates... 

A retrovirus with a nuclear localization signal can be used instead so that lacZ expression is restricted to the nucleus (18). This allows the cytoplasmic expression of other antigens to be detected more easily. 

Fig. 27.1. Efficiency of electroporation. The efficiency of in-ovo electroporation was checked by injecting the lacZ expression vector (pMiwZ) or the GFP expression vector (pEGFP-Nl) at stage 10. The lacZ expression is already recognizable 2h after the electroporation (A) and becomes strong 3h after the electroporation (B). Nine hours after the electroporation with GFP vector, one has the image in C. The efficiency of transfection can be checked in ovo with the GFP vector. At the transfection zone 24 h aftCT the electroporation (D and E), more than half of the cells express lacZ. The expression is transient, but lacZ expression is still strong 72 h after the electroporation (F). The lacZ transfection exats no morphological effects. The arrow in D indicates the section in E. Bars are 200 pm (A, B, C, D, F) 50pm(E). Source (3).

After incubation, fix and develop the explant coculture for lacZ expression in reporter cells as outlined in steps 14-19. 

After overnight incubation, remove the homogenates and RA-containing F9 assay medium from the cells, wash twice with PBS, and develop the reporter cells for detection of lacZ expression as described in Subheading 3.2., steps 14-19... 

After 4 d of growth at 30°C, the colonies that grow under histidine selection are assayed for lacZ expression using the -galactosidase filter assay described in Subheading 3.3.2. This protocol uses filter-paper circles that are 145 mm in diameter and these are incubated on 9 mL of Z-buffer containing X-gal dispensed in the lids of 150-mm culture dishes. 

This represents a 10-fold concentration of the original Hf7c culture, which may not be necessary in all cases. LacZ expression is strain-dependent, reflecting both the specific-promoter sequence driving the lacZ reporter and the strength of the interaction being examined. 

Wright N.J.D. and Zhong Y. 1995. Characterization of K+ currents and the cAMP-dependent modulation in cultured Drosophila mushroom body neurons identified by lacZ expression. J. Neurosci. 15 1025-1034. 

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