Background

The LacZ gene has been widely used as a reporter gene for detecting gene expression patterns (Stevens et al., 1989; Bonnerot and Nicolas, 1993). The protein encoded by the LacZ gene is β-galactosidase, which is able to produce visible color when incubated with specific substrates. Usually, LacZ is placed downstream of a target gene promoter in lieu of the coding sequence of the gene. Therefore, visualizing LacZ expression patterns models the endogenous expression pattern of the target gene (Figure 1A). The most popular LacZ staining approach is the X-gal/FeCN method, in which β-galactosidase catalyzes X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) hydrolysis into 5-bromo-4-chloro-3-hydroxyindole and galactose. Then, 5-bromo-4-chloro-3-hydroxyindole is oxidized into an insoluble blue dimer with the help of potassium ferricyanide and potassium ferrocyanide, which finally displays blue color (Figure 1B) (Pearson et al., 1963; Lojda, 1970). While the X-gal/FeCN staining method is highly specific and has low background, it is unable to detect genes expressed at low levels due to the low sensitivity of this stain (Gugliotta et al., 1992; Sundararajan et al., 2012). The S-gal/TNBT staining method was developed to study genes with low expression. S-gal (6-Chloro-3-indolyl-β-D-galactopyranoside), a chromogenic substrate like X-gal, can be hydrolyzed by β-galactosidase and TNBT (Tetranitro Blue Tetrazolium) to produce dark-brown formazan compounds under reducing conditions (Figure 1C). The S-gal/TNBT method is more sensitive than the X-gal/FeCN one, but a drawback is that it can have strong non-specific background staining (Sundararajan et al., 2012).

By combining both the methods described above, we developed an improved LacZ reporter staining protocol that is highly sensitive and highly specific. This method adds an additional staining step from the S-gal/TNBT method to the X-gal/FeCN method. In our staining technique, the first staining step, which is from the X-gal/FeCN method, creates an oxidative environment which consumes non-specific enzymatic activity; the second step, which is from the S-gal/TNBT method, is a reaction that is specific for β-galactosidase and results in a sensitive and specific signal. The improved method described here has been validated by detecting several genes in different embryo stages. Furthermore, this method has been used to study relatively highly expressed genes with good results: strong and specific staining, with slightly higher background than the X-gal/FeCN method (Shen et al., 2017).


Figure 1. Schematic diagram of LacZ reporters and -Galactosidase reactions. (A) The LacZ reporter gene is placed downstream of the target gene's promoter. Thus, when the expression of the target gene is induced, the gene product of LacZ, β-galactosidase, is produced. The key steps of the enzymatic reactions in the X-gal/FeCN (B) and S-gal/TNBT (C) staining methods.