Research reportAlteration of behavioural phenotype in mice by targeted disruption of the progranulin gene
Introduction
It is well established that the mammalian brain possesses sexually dimorphic structure, which is reflected by functional sexual differences in the control of specific types of behaviour and endocrine patterns [1]. In rodent species, such as rats and mice, sexual dimorphism of the brain is established during the perinatal period, which is known as the critical period for brain sexual differentiation. During this period, androgens secreted from the testis masculinize the brain in males, while the female brain is not exposed to androgens and thereby develops into the “default” female type. In the brain, androgens are converted to estrogens by aromatase and are then involved in the masculinization of the brain through the facilitation of transcription of various factors [32].
In an attempt to identify genes that are involved in sexual differentiation of the brain, we have isolated the progranulin (PGRN) gene as an androgen-inducible gene in neonatal rat hypothalamus [45]. The expression of PGRN mRNA in the hypothalamus of males is maintained at high levels throughout the critical period, yet abruptly declines in females after birth. As estrogens can induce PGRN gene expression in the hypothalamus [48], androgens probably induce PGRN gene expression after being converted to estrogens. We further observed that infusion of an antisense oligodeoxynucleotide for the PGRN gene into the neonatal male rat brain significantly suppressed male sexual behaviours after maturation [46], suggesting that the PGRN gene is indeed involved in the process of masculinization of the rat brain. Recently, we have also found that PGRN is involved in estrogen-induced neurogenesis in the adult rat hippocampus [11].
In the mouse, the PGRN gene spans approximately 6.3 kbp and contains 13 exons [3]. PGRN mRNA has been demonstrated in various tissues and organs including the reproductive organs, gastrointestinal tract, endocrinal organs and neural tissues [9], [17]. Peptides of approximately 6 kDa called granulins [6], [8] also known as epithelins [42] are derived from PGRN. Both PGRN and granulins have growth-modulating effects on many cell types in culture [7], [42]. For example, PGRN mediates the mitogenic effect of estrogens on MCF-7 cells, a human breast cancer cell line [30], which supports the above-mentioned notion that transcription of the PGRN gene is induced by estrogens. In addition, it has been recently reported that mutations in PGRN cause tau-negative, ubiquitin-positive frontotemporal dementia (FTD) in humans [4], [16], suggesting that PGRN is involved in neuronal survival.
To elucidate the physiological roles of PGRN/granulins in vivo, especially their roles in the brain, we generated mice lacking the PGRN gene in the present study. Here, we describe their phenotypes regarding sexually dimorphic behaviours, such as sexual behaviour, aggression and anxiety.
Section snippets
Generation of mice lacking the PGRN gene
A PGRN genomic clone was isolated from the 129 SvJ mouse genomic library (Stratagene, La Jolla, CA). The targeting vector was constructed by replacing an internal 4.7 kb EcoRV–EcoRI fragment, encoding exons 2–13 of the PGRN gene, with a PGK-Neo-bpA cassette [43] and by ligating the diphtheria toxin A (DT-A) fragment [54] to the 5′ end of the vector (Fig. 1A). The neomycin resistance gene, under the control of the phosphoglycerate kinase (PGK) 1 promoter, was inserted as a positive selection
Generation of mice lacking the PGRN gene
We cloned the mouse PGRN gene and disrupted it in an ES cell line derived from the 129 SvJ mouse strain (Fig. 1A). Six targeted clones out of 547 were identified by Southern blot analysis and 3 of these (6D5, 18C4 and 19B2) had normal karyotypes and were used for the production of chimeric mice. The chimeric mice were mated with C57BL/6 females. One of the chimeras, 6D5-#6 was shown to be a germ line chimera and heterozygous offspring obtained from him were backcrossed to C57BL/6 females.
Discussion
In the present study, we generated mice with targeted disruption of the PGRN gene to investigate the possible role of PGRN/granulins in the brain. Since it is reported that PGRN is expressed in the acrosome of the sperm [3] and oocytes [47] and that PGRN modulates the development of early embryos in vitro [19], we suspected that Grn−/− mice might be fetal lethal or exhibit disorders in reproductive function. However, they were fertile, and both litter size and the number of weaned pups were not
Acknowledgements
Y.K. and S.C. equally contributed to this work. This work was supported by Grants-in-Aid for Scientific Research (17208025, 17052003 to M.N.) from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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2021, Journal of Biological ChemistryCitation Excerpt :The Tmem106b−/− gene trap line is a hypomorph and expresses 5 to 10% residual full-length TMEM106B protein (76, 87). Grn−/− mice on a C57BL/6J background (RBRC02370; Kayasuga et al. (88)) were obtained from RIKEN BioResource Center and bred at our institution's animal facility. AppNL-F/WT heterozygous KI mice were imported from the RIKEN Institute.