(A) Structure of human chromosome 15.
(B) Schematic representation of the human 15q11-q13 region (not drawn to scale). The centromeric (cen) and telomeric (tel) positions are labeled. PWS protein coding genes and snoRNA genes are marked as boxes and ovals, respectively. Paternally, maternally, and biparentally expressed genes are labeled blue, red, and white, respectively. IPW exons of the paternally expressed, imprinted U-UBE3A-AS transcript are indicated by green bars and a blue arrow, respectively. The imprinting center (IC) is indicated with a small circle. The snoRNA genes: HBII-436, HBII-13, HBII-437, HBII-438A, HBII-85. HBII-52 and HBII-438B are labeled as 436, 13, 437, 438A, 85, 52 and 438B, accordingly.
(C) Corresponding mouse syntenic chromosome 7C region. The paternally expressed Lncat transcript is indicated by a blue arrow.
(D) Deletions in the PWS/AS region. (1) Insertion of the Epstein–Barr virus Latent Membrane Protein 2A, (LMP2A) transgene into the PWS/AS locus resulted in deletion of approximately 4 megabases between the Mlsn1 and Gas2 genes [,]. Depending on the parental origin it causes PWS or AS symptoms in mice. (2) The 42 kb Snrpn promoter PWS-IC deletion resulted in loss of imprinting and a PWS-like phenotype in mice []; analogous deletion of the PWS-IC and Snrpn gene up to exon 7 results in a paternal to maternal imprint switch and leads to PWS []. (3) Paternal Snrpn exon 2 deletion [] produced no abnormal phenotype. (4) Mice with a deletion of 1kb (exons 5–7) of the Snrpn gene are phenotypically normal []. (5) Paternal deletion from Snrpn to Ube3a resulted in a PWS-like phenotype in mice []. (6) Deletion affecting npc Ipw exons and MBII-52 snoRNA gene cluster after paternal inheritance produced no PWS-like phenotype []. (7) Targeting of the Necdin gene produced either a PWS-like phenotype [,] or no obvious phenotype []. The discrepancies were explained by different strategies in Necdin targeting and different mouse genetic backgrounds []. (8) Inactivation of the Magel2 gene produced altered behavioral rhythmicity []. (9) Deletion of the Mkrn3 (Zfp127) gene had no apparent phenotypic effect []. (10) PWS critical region (PWScr). It has been proposed that deletion of this region should play a major role in the development of PWS [,]. Paternal deletion of PWScr causes growth retardation in mice (this study).
(E) Locus containing the cluster of MBII-85 snoRNA genes and Ipw exons, and its deletion targeting strategy using “chromosome engineering” []. Filled boxes represent 5′HR and 3′HR DNA probes (labeled green and purple respectively). Targeting constructs 5′HPRT/PWScr_targ and 3′HPRT/PWScr_targ are indicated. Open boxes correspond to the Thymidine kinase gene (TK), Neomycin resistance gene (Neo), Puromycin resistance gene (Puro), as well as the 5′ and 3′ parts of the Hypoxanthine Phosphoribosyl Transferase gene (HPRT). LoxP sites are indicated by arrows.
(F) Structure of the PWScr after two consecutive HR events, and CRE recombinase-induced deletion.
(G) Sequencing of integration sites after HR and deletion of the PWScr confirmed the anticipated deletion at the nucleotide level. Extended sequences (Genbank accession number EU233428) of regions flanking the HPRT cassette insertion are presented in .