Generation and characterization of telomere length maintenance in tankyrase 2-deficient mice

Abstract

Telomere length and function are crucial factors that determine the capacity for cell proliferation and survival, mediate cellular senescence, and play a role in malignant transformation in eukaryotic systems. The telomere length of a specific mammalian species is maintained within a given range by the action of telomerase and telomere-associated proteins. TRF1 is a telomere-associated protein that inhibits telomere elongation by its binding to telomere repeats, preventing access to telomerase. Human TRF1 interacts with tankyrase 1 and tankyrase 2 proteins, two related members of the tankyrase family shown to have poly(ADP-ribose) polymerase activity. Human tankyrase 1 is reported to ADP-ribosylate TRF1 and to down-regulate the telomeric repeat binding activity of TRF1, resulting in telomerase-dependent telomere elongation. Human tankyrase 2 is proposed to have activity similar to that of tankyrase 1, although tankyrase 2 function has been less extensively characterized. In the present study, we have assessed the in vivo function of mouse tankyrase 2 by germ line gene inactivation and show that inactivation of tankyrase 2 does not result in detectable alteration in telomere length when monitored through multiple generations of breeding. This finding suggests that either mouse tankyrases 1 and 2 have redundant functions in telomere length maintenance or that mouse tankyrase 2 differs from human tankyrase 2 in its role in telomere length maintenance. Tankyrase 2 deficiency did result in a significant decrease in body weight sustained through at least the first year of life, most marked in male mice, suggesting that tankyrase 2 functions in potentially telomerase-independent pathways to affect overall development and/or metabolism.

FIG.1.

Generation of TANK2-deficient mice by gene targeting. (A) Gene targeting strategy and restriction map of TANK2 gene. Filled boxes indicate exons, and labeled boxes indicate neomycin (neo) resistance or herpesvirus thymidine kinase (tk) genes. loxP sites are indicated. (B) Southern blot analysis of ES cell DNA. The 8.5-kb band represents the germ line allele. The 5.4- and 4-kb bands represent the targeted alleles. (C) PCR analysis for the conditional TANK2 knockout mouse genotype. The 270- and 340-bp PCR products represent the wild-type and floxed alleles, respectively. (D) PCR analysis for the constitutive TANK2 knockout mouse genotype. The 270- and 240-bp PCR products represent the wild-type and knockout alleles, respectively. (E) RT-PCR analysis for TANK2 mRNA expression. 5′-TANK2 and 3′-TANK2 PCR products represent upstream and downstream cDNA of TANK2, respectively. Actin cDNA was the RT-PCR loading control.

FIG.1.

Generation of TANK2-deficient mice by gene targeting. (A) Gene targeting strategy and restriction map of TANK2 gene. Filled boxes indicate exons, and labeled boxes indicate neomycin (neo) resistance or herpesvirus thymidine kinase (tk) genes. loxP sites are indicated. (B) Southern blot analysis of ES cell DNA. The 8.5-kb band represents the germ line allele. The 5.4- and 4-kb bands represent the targeted alleles. (C) PCR analysis for the conditional TANK2 knockout mouse genotype. The 270- and 340-bp PCR products represent the wild-type and floxed alleles, respectively. (D) PCR analysis for the constitutive TANK2 knockout mouse genotype. The 270- and 240-bp PCR products represent the wild-type and knockout alleles, respectively. (E) RT-PCR analysis for TANK2 mRNA expression. 5′-TANK2 and 3′-TANK2 PCR products represent upstream and downstream cDNA of TANK2, respectively. Actin cDNA was the RT-PCR loading control.

FIG. 2.

Telomere length was not altered in TANK2 knockout mice. (A) Spleen cells were isolated from TANK2^+/+ (n = 7), TANK2^−/−G1 (n = 7), and TANK2^−/−G5/G7 (n = 7) mice, and relative telomere length was determined by Flow-FISH. The fluorescein isothiocyanate fluorescent signal of the cell binding telomeric probe was converted to arbitrary units of molecule equivalents of soluble fluorescence (MESF). (B and C) Histograms, based on Q-FISH data from the comparison of one TANK2^+/+ mouse with one TANK2^−/−G1 mouse (B) and the comparison of one TANK2^+/+ mouse with one TANK2^−/−G7 mouse (C), depict the distribution of fluorescent intensities corresponding to individual telomeres of different lengths present in metaphase chromosomes. (D) Spleen cells were isolated from TANK2^+/+ (n = 8), TANK2^−/−G1 (n = 8), and TANK2^−/−G5/G7 (n = 9) mice, and relative telomere length was determined by Q-FISH. The average of fluorescent intensities from each mouse was normalized to that of a TANK2^+/+ mouse (defined as 100). The relative telomere length of each strain of mice is plotted.

FIG. 3.

The body weights of tankyrase 2 knockout mice were reduced. (A) Body weight changes in female TANK^+/+ (n = 5) and TANK^−/− (n = 6) mice. (B) Body weight changes in male TANK^+/+ (n = 6) and TANK^−/− (n = 7) mice.