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Ectopic Expression of Retrotransposon-Derived PEG11/RTL1 Contributes to the Callipyge Muscular Hypertrophy.

Xu X, Ectors F, Davis EE, Pirottin D, Cheng H, Farnir F, Hadfield T, Cockett N, Charlier C, Georges M, Takeda H - PLoS ONE (2015)

Bottom Line: The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous +Mat/CLPGPat animals receiving the CLPG mutation from their father express the phenotype. +Mat/CLPGPat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle.We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype.Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep.

View Article: PubMed Central - PubMed

Affiliation: Unit of Animal Genomics, GIGA Research Center and Faculty of Veterinary Medicine, University of Liège, 1 Avenue de l'Hôpital, Liège, Belgium.

ABSTRACT
The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous +Mat/CLPGPat animals receiving the CLPG mutation from their father express the phenotype. +Mat/CLPGPat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle. We showed previously in transgenic mice that ectopic expression of DLK1 alone induces a muscular hypertrophy, hence demonstrating a role for DLK1 in determining the callipyge hypertrophy. We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype. Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep.

No MeSH data available.


Related in: MedlinePlus

Characterization of the oPEG11 transgene.A. Organization of the oPEG11 transgene (TP) integration site in mouse oPEG11 line 126 and endogenous mouse Myosin light chain (Mlc) locus. Thick black lines correspond to upstream, intronic and downstream sequences of the Mlc gene including the 3F promotor (Mlc3F) and 2E enhancer (Mlc2E); the numbered black boxes to Mlc exons; the thick grey line to vector-specific sequences including a 220 bp duplicated segment marked by a small white box (dup); the light gray boxes to the open reading frame of ovine PEG11 (oPEG11 ORF) and bovine GH polyadenylation site (pA), respectively; the thin black lines to the transgene integration site flanked by Robo2 at 407 Kb on the proximal site, and Lipi at 722 Kb on the distal site. The positions of the NotI and SmaI sites in the vector intended to excise the insert DNA for microinjection are shown, as are the positions of the SpeI and MfeI sites determining the size of the restriction fragments detected with the indicated probe (probe) in Southern blotting for the transgene integration site and endogenous Mlc gene. The position of greater than or equal to 1.6 Kb deletion at the integration site is indicated (del). The position of the primers used for PCR and RT-PCR are shown. B. Results of Southern blotting of genomic DNA of homozygous +/+, heterozygous +/TP, and homozygous TP/TP mice (oPEG11 line 126), digested with SpeI (left) or SpeI + MfeI (right), using the probe with position as shown in A. The bands corresponding to the transgene (TP), and endogenous Mlc gene (mMlc) are marked. C. Results of PCRs performed using genomic DNAs and the primers shown in A to clarify the transgene organization in mouse oPEG11 lines 126 and 127A.
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pone.0140594.g001: Characterization of the oPEG11 transgene.A. Organization of the oPEG11 transgene (TP) integration site in mouse oPEG11 line 126 and endogenous mouse Myosin light chain (Mlc) locus. Thick black lines correspond to upstream, intronic and downstream sequences of the Mlc gene including the 3F promotor (Mlc3F) and 2E enhancer (Mlc2E); the numbered black boxes to Mlc exons; the thick grey line to vector-specific sequences including a 220 bp duplicated segment marked by a small white box (dup); the light gray boxes to the open reading frame of ovine PEG11 (oPEG11 ORF) and bovine GH polyadenylation site (pA), respectively; the thin black lines to the transgene integration site flanked by Robo2 at 407 Kb on the proximal site, and Lipi at 722 Kb on the distal site. The positions of the NotI and SmaI sites in the vector intended to excise the insert DNA for microinjection are shown, as are the positions of the SpeI and MfeI sites determining the size of the restriction fragments detected with the indicated probe (probe) in Southern blotting for the transgene integration site and endogenous Mlc gene. The position of greater than or equal to 1.6 Kb deletion at the integration site is indicated (del). The position of the primers used for PCR and RT-PCR are shown. B. Results of Southern blotting of genomic DNA of homozygous +/+, heterozygous +/TP, and homozygous TP/TP mice (oPEG11 line 126), digested with SpeI (left) or SpeI + MfeI (right), using the probe with position as shown in A. The bands corresponding to the transgene (TP), and endogenous Mlc gene (mMlc) are marked. C. Results of PCRs performed using genomic DNAs and the primers shown in A to clarify the transgene organization in mouse oPEG11 lines 126 and 127A.

Mentions: We combined Southern blotting, regular PCR, and “splinkerette” PCR [17] to characterize the transgene insertions in mouse lines 126 and 127. Line 126 was shown to harbor a single-copy transgene that was integrated into an intergenic region on chromosome 16, at ∼407 Kb from Roundabout homolog 2 (Robo2) and ∼722 Kb from Lipase, member I (Lipi) (Fig 1; S1 Fig). Unexpectedly, the transgene comprised both the Mlc/oPEG11 insert, as well as the remainder of the vector including a 0.22 Kb duplicated segment flanking the transgene on either end (Fig 1). This indicated that the microinjected insert preparation was contaminated with undigested vector. The restriction patterns obtained by Southern blotting suggest that the transgene insertion was accompanied by a more than or equal to 1.6 Kb deletion at the integration site (Fig 1).


Ectopic Expression of Retrotransposon-Derived PEG11/RTL1 Contributes to the Callipyge Muscular Hypertrophy.

Xu X, Ectors F, Davis EE, Pirottin D, Cheng H, Farnir F, Hadfield T, Cockett N, Charlier C, Georges M, Takeda H - PLoS ONE (2015)

Characterization of the oPEG11 transgene.A. Organization of the oPEG11 transgene (TP) integration site in mouse oPEG11 line 126 and endogenous mouse Myosin light chain (Mlc) locus. Thick black lines correspond to upstream, intronic and downstream sequences of the Mlc gene including the 3F promotor (Mlc3F) and 2E enhancer (Mlc2E); the numbered black boxes to Mlc exons; the thick grey line to vector-specific sequences including a 220 bp duplicated segment marked by a small white box (dup); the light gray boxes to the open reading frame of ovine PEG11 (oPEG11 ORF) and bovine GH polyadenylation site (pA), respectively; the thin black lines to the transgene integration site flanked by Robo2 at 407 Kb on the proximal site, and Lipi at 722 Kb on the distal site. The positions of the NotI and SmaI sites in the vector intended to excise the insert DNA for microinjection are shown, as are the positions of the SpeI and MfeI sites determining the size of the restriction fragments detected with the indicated probe (probe) in Southern blotting for the transgene integration site and endogenous Mlc gene. The position of greater than or equal to 1.6 Kb deletion at the integration site is indicated (del). The position of the primers used for PCR and RT-PCR are shown. B. Results of Southern blotting of genomic DNA of homozygous +/+, heterozygous +/TP, and homozygous TP/TP mice (oPEG11 line 126), digested with SpeI (left) or SpeI + MfeI (right), using the probe with position as shown in A. The bands corresponding to the transgene (TP), and endogenous Mlc gene (mMlc) are marked. C. Results of PCRs performed using genomic DNAs and the primers shown in A to clarify the transgene organization in mouse oPEG11 lines 126 and 127A.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608697&req=5

pone.0140594.g001: Characterization of the oPEG11 transgene.A. Organization of the oPEG11 transgene (TP) integration site in mouse oPEG11 line 126 and endogenous mouse Myosin light chain (Mlc) locus. Thick black lines correspond to upstream, intronic and downstream sequences of the Mlc gene including the 3F promotor (Mlc3F) and 2E enhancer (Mlc2E); the numbered black boxes to Mlc exons; the thick grey line to vector-specific sequences including a 220 bp duplicated segment marked by a small white box (dup); the light gray boxes to the open reading frame of ovine PEG11 (oPEG11 ORF) and bovine GH polyadenylation site (pA), respectively; the thin black lines to the transgene integration site flanked by Robo2 at 407 Kb on the proximal site, and Lipi at 722 Kb on the distal site. The positions of the NotI and SmaI sites in the vector intended to excise the insert DNA for microinjection are shown, as are the positions of the SpeI and MfeI sites determining the size of the restriction fragments detected with the indicated probe (probe) in Southern blotting for the transgene integration site and endogenous Mlc gene. The position of greater than or equal to 1.6 Kb deletion at the integration site is indicated (del). The position of the primers used for PCR and RT-PCR are shown. B. Results of Southern blotting of genomic DNA of homozygous +/+, heterozygous +/TP, and homozygous TP/TP mice (oPEG11 line 126), digested with SpeI (left) or SpeI + MfeI (right), using the probe with position as shown in A. The bands corresponding to the transgene (TP), and endogenous Mlc gene (mMlc) are marked. C. Results of PCRs performed using genomic DNAs and the primers shown in A to clarify the transgene organization in mouse oPEG11 lines 126 and 127A.
Mentions: We combined Southern blotting, regular PCR, and “splinkerette” PCR [17] to characterize the transgene insertions in mouse lines 126 and 127. Line 126 was shown to harbor a single-copy transgene that was integrated into an intergenic region on chromosome 16, at ∼407 Kb from Roundabout homolog 2 (Robo2) and ∼722 Kb from Lipase, member I (Lipi) (Fig 1; S1 Fig). Unexpectedly, the transgene comprised both the Mlc/oPEG11 insert, as well as the remainder of the vector including a 0.22 Kb duplicated segment flanking the transgene on either end (Fig 1). This indicated that the microinjected insert preparation was contaminated with undigested vector. The restriction patterns obtained by Southern blotting suggest that the transgene insertion was accompanied by a more than or equal to 1.6 Kb deletion at the integration site (Fig 1).

Bottom Line: The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous +Mat/CLPGPat animals receiving the CLPG mutation from their father express the phenotype. +Mat/CLPGPat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle.We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype.Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep.

View Article: PubMed Central - PubMed

Affiliation: Unit of Animal Genomics, GIGA Research Center and Faculty of Veterinary Medicine, University of Liège, 1 Avenue de l'Hôpital, Liège, Belgium.

ABSTRACT
The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous +Mat/CLPGPat animals receiving the CLPG mutation from their father express the phenotype. +Mat/CLPGPat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle. We showed previously in transgenic mice that ectopic expression of DLK1 alone induces a muscular hypertrophy, hence demonstrating a role for DLK1 in determining the callipyge hypertrophy. We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype. Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep.

No MeSH data available.


Related in: MedlinePlus