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Determination of lacZ transgene expression in the mouse brain done on razor-made-slices, vibratome, cryo- and paraffin sections

Transgene lacZ is a common part of the various DNA constructs used to genetically modify mice either via oocyte injection or embryonic stem cell transfection. The activity of lacZ can be driven by the inserted promoter or by an endogenous promoter of the gene in which lacZ was located. After transcription lacZ mRNA is translated on the ribosomes in beta-galactosidase protein. This protein is not present in the eukaryotic cells, hence it can serve as an excellent marker in transgenic animals. Beta-galactosidase can act on several substrates, from which the most common is X-gal (C14H15BrClNO6, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Beta-galactosidase changes the colorless substrate in the blue staining, which is stable in the dark, it does not diffuse in the surrounding tissue and it can be easily visualized with the aid of the classical light microscope (1). To assess the expression of the genes modified by gene trap, the gene trap vector was designed to contain lacZ gene. lacZ was present in the construct without its own promoter, preceded only with a splice acceptor. The gene trap vector was used to modify mouse embryonic stem cells and after selection, the appropriate clones of embryonic stem cells were used to generate a mouse carrying the corresponding genetic modification. Due to the nature of the inserted construct lacZ was active only if it was placed within the endogenous gene. The promoter of in such way “ trapped” endogenous gene was responsible for lacZ transcription. lacZ was considered as an exon due to the presence of the splice acceptor, and it was correctly translated only if it was in frame with the “ trapped” endogenous gene (2). Mouse line Stam2Gt1Gaj, containing lacZ in frame with Stam2 gene was used for this investigation (3). The resulting beta-galactosidase activity was assessed in the brain of the heterozygous and homozygous adult carriers of gene trap mutation. The purpose of the work was to establish the reliable methods of assessing the beta-galactosidase activity by histochemical X-gal staining in the mouse tissues. In case the tissue sample is thick the amount of the signal is higher and easier to detect, nevertheless its localization is rather obscure. Therefore the histochemical staining was performed on brain samples prepared as razor-made-slices, vibratome, cryo- and paraffin sections. Razor-made-slices of the mouse brain were made by a home-made device containing razor blades separated by 2mm gaps. The animals were fixed by perfusion with 2% formaldehyde and 0.2% glutaraldehyde. The brain was isolated and cut by the arranged razor blades. The sectioning was facilitated by the fact that the tissue was hardened by fixative used for perfusion. The brain slices were fixed further for 1 hour in the same fixative and washed by phosphate buffered saline (PBS) containing detergents, 0.01% Igepal (Sigma), 0.01% Na-deoxycholate (Kemika). After extensive rinsing, the slices were transferred to staining solution: 1mg/ml X-gal (Merck), 2 mM MgCl2, 10 mM K4Fe(Cn)6, 10 mM K3Fe(Cn)6, 0.01% Igepal and 0.01% Na-deoxycholate in PBS for 24 hours on 37°C on the shaker in the dark. The stained slices were rinsed in PBS and cleared in ascending concentrations of glycerol up to 70% and visualized by stereomicroscope (fig. 1). The vibratome sections were made on brains isolated form perfusion fixed animals as above. The brain was subjected to additional 30 min immersion fixation before the sectioning in order to harden the brain enough to be sectioned. The brain was attached to the holder by the instant glue SuperAtack (Loctite). The vibratome sections were 50-200 µ m thick, they were collected in 6-well plates in PBS with detergents for rinsing, and afterwards transferred to the staining solution in the same conditions as above (Fig. 2). For cryo-sections the brains were isolated from perfusion fixed animals, subjected to immersion fixation for 30 minutes, cryoprotected with 10% sucrose for 24 hours. The brains were transferred in isopentane on -80°C for 1 minute and stored in polypropilene tubes on -80°C. Before sectioning the brains were fixed on holder with Tissue Freezing Medium (Jung). The 35 µ m sections were made on cryotome and transferred to PBS in 6-well plates. Using this free-floating method the sections were rinsed with PBS and transferred to staining solution (Fig. 3). Paraffin technique was also tried, but the staining was not possible on paraffin embedded sections. This technique could be eventually used for the pre-stained whole mount specimens (e.g. embryos). The presented methods are complementary and could be used together to appropriately localize the presence of beta-galactosidase. The razor-blade slices are easy to obtain and give an overwiev of the expression, the vibratome sections give precise location of staining in different parts of the brain, while the cryosections show the localization of the staining in the cells or even within the cells.

lacZ; expression pattern; brain; mouse

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