Characterization of transgenic mice with overexpression of spermidine synthase

Abstract

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.

Figure 1. Transgene construct and PCR identification of transgenic mice

A, The human SpdS cDNA was placed under the control of composite cytomegalovirus-immediate early gene enhancer/chicken β-actin (CAG) promoter elements to generate the transgene expression construct. An HA epitope was added to the amino terminus of the hSpdS to enable detection of transgene-derived SpdS protein. Arrows indicate the positions of primers used for PCR identification of transgenic mice in B. B, A representative analytic gel of tail DNA extracts from eight potential transgenic mice (lanes 2 to 9) and a DNA standard (lane 1). Genotyping by PCR yielded a 397 bp product from the genomic DNA from transgenic animals (lanes 2, 7 and 9). Primers that amplify a 520 bp product from the mouse antizyme gene were included in all reactions as a positive control.

Figure 2. Detection of SpdS protein in tissue extracts from CAG-SpdS mice

Tissues extracts of skeletal muscle, heart and liver from wild type (wt) control and transgenic mice from each founder line (A and B) were blotted for SpdS protein using anti-hSpdS antibody (top) or anti-HA antibody (bottom). Each lane contains 50 μg of cytosolic proteins.