Tissue-specific inactivation of murine M6P/IGF2R

Abstract

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and T cell- mediated immunity. M6P/IGF2R is an imprinted gene in mice with expression only from the maternal allele. Complete knockout of this gene causes neonatal lethality, thus preventing analysis of its multifunctional role postnatally. To help elucidate the biological functions of M6P/IGF2R in adulthood, we generated both complete and tissue-specific M6P/IGF2R knockout mice using the Cre/loxP system. We confirm that complete M6P/IGF2R knockout results in fetal overgrowth and neonatal lethality. In contrast, tissue-specific inactivation of this gene in either the liver or skeletal and cardiac muscle gives rise to viable animals with no obvious phenotype. The successful creation of viable tissue-specific M6P/IGF2R knockout mouse models will now allow for detailed analysis of receptor function in a number of cellular processes including brain development, carcinogenesis, lysosomal trafficking, and T cell-mediated immunity.

Figure 1.

LoxP targeting of M6P/IGF2R exon 10. A: Generation of the M6P/IGF2R LoxP targeting construct involved the cloning of 7-kb of the mouse genomic DNA encompassing exons 8 through 11 and flanking intronic sequence into an appropriate LoxP vector. The M6P/IGF2R LoxP targeting construct was then transfected into Sv129 ES cells. Homologous recombinants were selected for using G418 (neomycin) and confirmed by PCR. B: Cre-recombinase plasmid (1 μg) controlled from the constitutively active CMV promoter was transfected into the ES cells which were then grown in the presence of gancyclovir to select for recombinants that had lost the TK/Neo cassette; PCR was used to confirm loss of the TK/Neo cassette. Recombinant ES cells were injected into the blastocoele cavity of the 3.5-day-old C57Bl/6J embryos before implantation into pseudo-pregnant mice. C: Expression of Cre-recombinase in mice with a floxed exon 10 results in its excision generating a M6P/IGF2R protein that encodes for a truncated receptor lacking the M6P and IGF2 extracellular binding domains. DT, TK, and Neo enable both positive and negative selection.

Figure 2.

Cre/loxP strategy successfully creates a functionally null M6P/IGF2R allele. A: Wild-type (WT) and M6P/IGF2R knockout (KO) mice at 18.5 days of gestation. Embryos inheriting a deleted M6P/IGF2R exon 10 allele from their mothers (KO) exhibited an overgrowth phenotype. They also frequently displayed extra post-axial digits on the fore and/or hind limbs (arrowhead); shorter, blunter tails often with a characteristic kink in the tip (*); and extravasation of overgrown abdominal organs (arrow). B: Wet weight measurements demonstrate that knockout (KO) embryos (n = 12) and their corresponding placentas are significantly (P < 0.0001) larger than those in WT mice (n = 20); standard errors of the mean are shown. C: Western blot analysis performed on tail tips from the 18.5-day-old embryos confirmed the absence of M6P/IGF2R protein in KO mice.

Figure 3.

M6P/IGF2R immunohistochemical staining of lung at 18.5 days of gestation. A: Wild-type mouse lung, B: M6P/IGF2R knockout mouse lung, and C: M6P/IGF2R knockout mouse lung with reactivated receptor expression. Arrowheads; bronchial alveolar structures with expression of the normally silenced paternal M6P/IGF2R allele.

Figure 4.

Western blot analysis of tissue-specific M6P/IGF2R knockout mice. A: Protein extracts were prepared from tissues of M6P/IGF2R liver-specific knockout (KO) (n = 5) and wild-type (WT) (n = 5) animals; representative samples are shown. 100 μg of total protein from each tissue was electrophoresed in a NuPAGE 4 to 12% gradient polyacrylamide gel, blotted onto nitrocellulose filters, and probed with a polyclonal M6P/IGF2R antibody. B: Protein extracts were prepared from tissues removed from M6P/IGF2R skeletal/cardiac KO (n = 5) and WT (n = 5) animals; representative samples are shown. 100 μg of total protein from each tissue was electrophoresed in a NuPAGE 4 to 12% gradient polyacrylamide gel, blotted onto nitrocellulose filters, and probed with a polyclonal M6P/IGF2R antibody. The blot labeled “Muscle” was exposed longer than the other tissues to reveal the faint band in the KO lane.