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. 2023 Feb 3;16(2):dmm049829. doi: 10.1242/dmm.049829

Fig. 1.

Fig. 1.

Strategy and validation of a novel Magel2 rat model. (A) Diagram showing the overall strategy to create a Magel2 rat model. Rat Magel2: single-exon gene located on chromosome 1 (genomic locus, white bar; 3777 bp), protein product [wild-type protein, white bar; 1258 amino acids (aa)] with a MAGE homology domain (MHD; green bar). Transcription activator-like effector nuclease (TALEN) modification of the wild-type locus created an 8 bp deletion (c.735_742; yellow circle), predicted to shift the open-reading frame and result in loss of full-length MAGEL2 protein (targeted protein, p.Ser132Glyfs728). Additional predicted features of the 8 bp-induced mutation include translational of a novel aa sequence (targeted protein, yellow bar) and loss of the conserved MHD. (B) Relative Magel2 mRNA abundance measured by qRT-PCR showed no difference in Magel2 expression levels in mutant genotype groups compared to wild-type littermate rats. Allelic inheritance and corresponding genotype of each Magel2 group is shown as either maternal (m) or paternal (p), and wild-type (+) or mutant (−). Hypothalamic tissue samples from rats heterozygous for the paternally or maternally inherited mutant Magel2 allele (m+/p− or m−/p+, respectively), rats homozygous for both mutant alleles (m−/p−) and wild-type rats (m+/p+) were used. Data are depicted as mean±s.e.m.; individual data points are denoted by black squares. n=3-6/rats per genotype group. (C) Endpoint RT-PCR and sequencing of hypothalamic cDNA from parental rat strains [maternal Sprague-Dawley (SD); paternal Long-Evans (LE)] and F1 intercross hybrid progeny (F1.SD.LE) confirms a parent-of-origin effect on rat Magel2 expression. The wild-type Magel2 coding sequence contains a single-nucleotide polymorphism (SNP) at position c.864 that differs between SD (c.864G) and LE (c.864A) rats. Representative sequence traces of the SNP from parental and F1 hybrid genotypes is shown (yellow highlight); F1(SD.LE) hybrid rats show expression of paternal LE-derived SNP, c.864A. Rat images created with BioRender.com. (D) Endpoint RT-PCR and sequencing of hypothalamic cDNA from rats derived from the Magel2 mutant model validate a parent-of-origin effect on rat Magel2 expression in the context of the TALEN-modified Magel2 rat allele (wild-type littermates, m+/p+; heterozygous Magel2 rats, m+/p− and m−/p+; homozygous rats, m−/p−). Representative sequence traces show each Magel2 genotype sequence aligned to the rat reference sequence. Matching sequences (dots) in each window are denoted. m+/p+ and m−/p+ show complete alignment. The absence of sequence (dash, highlighted in yellow) corresponding to the TALEN-generated 8 bp deletion was found only in Magel2 genotype groups with paternal inheritance of the mutation (m+/p− and m−/p−).