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. 1999 Aug 3;96(16):9258-63.
doi: 10.1073/pnas.96.16.9258.

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes

J M Gabriel  1 , M Merchant, T Ohta, Y Ji, R G Caldwell, M J Ramsey, J D Tucker, R Longnecker, R D Nicholls
Affiliations

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes

J M Gabriel et al. Proc Natl Acad Sci U S A. .

Abstract

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

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Figures

Figure 1
Figure 1
Imprinted phenotype (a) and inheritance (b) of TgPWS/AS(del) mice. (a) Representative 5-day-old littermates in which the wild-type animal (albino coat color) on left weighs 6.2 g, whereas a PWS pup inheriting the transgene paternally (dark coat color) weighs only 2.3 g. (b) Filled symbols represent the severe failure-to-thrive phenotype seen in transgenic offspring after paternal inheritance only (PWS model), whereas half-filled symbols represent animals inheriting the transgene mutation maternally (a genetic model of AS). This represents only one branch of the complete pedigree. The number of animals of a particular genotype in a given litter are indicated. Diamonds show animals of both or unidentified sex.
Figure 2
Figure 2
DNA methylation analysis of the Epstein–Barr virus LMP2A transgene. Paternal inheritance results in transgene DNA that is largely undigested by the methylation-sensitive restriction enzyme HpaII, whereas maternal inheritance of the transgene results in DNA that is mostly digested by HpaII (the 0.4- and 1.2-kb lower bands are unmethylated). Symbols are as for Fig. 1.
Figure 3
Figure 3
Expression analyses by RT-PCR. The nonimprinted control gene Herc2 is expressed in all animals (a and g). Mice inheriting the transgene paternally [Tg(P)] show a complete lack of expression of any of the five known paternally expressed genes in chromosome 7C (b–g), whereas wild-type littermates (WT) and mice inheriting the transgene maternally [Tg(M)] express each of these imprinted genes (total brain RNA was used for a–f and cerebellum RNA for g). In contrast, the Ube3a gene shows virtually no expression in cerebellum from Tg(M) mice compared with abundant expression in Tg(P) and WT cerebellum (g). A + or − indicates that reverse transcriptase was or was not added to the RT reaction, respectively.
Figure 4
Figure 4
DNA methylation analyses. (a) Digestion of DNA with EcoRI and MluI and hybridization with a Snurf–Snrpn exon 1 probe identifies bands corresponding to the methylated maternal (M) and unmethylated paternal (P) alleles in wild-type mice. Paternal or maternal inheritance of the transgene yields only the maternal or paternal methylation pattern, respectively. (b) Similar results are seen for Zfp127 (XbaI–EagI), mapping 1 megabase distal of Snrpn.
Figure 5
Figure 5
Mapping the extent of the deletion by FISH. (a) Metaphase chromosome spreads of mice heterozygous for the transgene were hybridized with a mouse chromosome 7 centromere-specific probe (also hybridizes to telomere of chromosome 5) coupled with the Snurf–Snrpn BAC 397F16. The single set of red signals indicates that this locus is deleted in transgenic animals. Similar FISH analysis demonstrates that the deletion encompasses Zfp127 (green signal) (b), BAC BH6 located just proximal of Ube3a (green signal) (c), and the 3′ end of the Herc2 gene (green signal) (d). In each case, the test probe is deleted from one chromosome 7 homolog. In contrast, two sets of green signals are seen for the Mlsn1 BAC 220N6 (e) and the Igf1r gene (f), indicating that these two loci are intact on the TgPWS/AS(del) chromosome. (g) Summary map showing that the deletion (thick line) associated with transgene insertion encompasses the imprinted and nonimprinted domains of mouse chromosome 7C that are syntenic to the PWS/AS-homologous domain in human 15q11–q13. Loci immediately proximal (Mlsn1), syntenic to human 15q13–q14, and distal (Igf1r), syntenic to human 15q25–qter, are intact. Dashed lines, translocations; cen, centromere; tel, telomere; sub, subclones; hatched boxes, FISH probes; + or −, intact or deleted FISH probe; blue or red, paternal- or maternal-only.
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