B6.Cg-Pabpn1^tm1.1Gpvl/J

Stock number: 029236
Product name: Pabpn1 conditional knock-in
Research areas: Neurobiology Research, Research Tools

Description

Pabpn1 conditional knock-in mice possess a 17-alanine (17) expanded Pabpn1 gene. Expression of this knock-in is prevented by an upstream floxed sequence containing a WT mouse Pabpn1 gene and an EGFP sequence. Cre Recombinase excision of the floxed region results in the expression of Pabpn1^A17, associated with the onset of Oculopharyngeal muscular dystrophy (OPMD).

Detailed description

Pabpn1 conditional knock-in mice possess loxP sites flanking a WT mouse poly(A) binding protein, nuclear 1 (Pabpn1) cDNA, an internal ribosomal entry site (IRES), and an enhanced green fluorescent protein (EGFP) sequence, preventing expression of a downstream 17 alanine-expanded endogenous Pabpn1 gene. PABPN1 is a ubiquitously expressed RNA binding protein that is most well characterized as a regulator of polyadenosine (poly[A]) tail length and alternative polyadenylation (APA), as well as a regulator of expression of antisense RNAs associated with gene promoters. A dominant expansion of ten amino terminal alanine residues to 11-18 alanines in PABPN1 is associated with the onset of Oculopharyngeal muscular dystrophy (OPMD). This is a late onset disease that causes ptosis, dysphagia, and loss of mobility due to progressive weakness of eyelid, pharyngeal, and proximal limb muscles. Patients are affected by loss of nutrition and aspiration pneumonia. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have the floxed sequence removed, allowing for expression of Pabpn1^A17 in cre-expressing tissues. Mice that are homozygous for this allele are viable and fertile.

When bred to B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (Stock No. 003724) mice, with early embryonic Cre Recombinase expression, resulting Pabpn1^A17 mice exhibit mild myopathic phenotype in adult and aged animals. They have shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage as noted by increased mitochondrial genome copy number. They also display hind limb clasping starting at nine months of age, and they have smaller hind limb muscles.

Development

The Pabpn1 conditional knock-in targeting vector was designed to insert a floxed sequence containing a WT mouse poly(A) binding protein, nuclear 1 (Pabpn1) cDNA, an internal ribosomal entry site (IRES), an enhanced green fluorescent protein (EGFP) sequence, and a frt-flanked neomycin resistance (neo) cassette. This floxed cassette was inserted upstream of the endogenous Pabpn1 sequence. Downstream of the second loxP site, a start codon is followed by an extra 7 alanines ((GCG)₇), added to the 10 endogenous alanines after the initial methionine, creating a 17 alanine-expanded Pabpn1 gene (A17 PABPN1). The construct was electroporated into B6(Cg)-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and chimeric males were bred with C57BL/6J females. Resulting offspring were bred to Flp-expressing mice to remove the neo cassette. Pabpn1 conditional knock-in mice were bred to remove the Flp-expressing transgene, and offspring were bred to C57BL/6J mice for at least 5 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Pabpn1^tm1.1Gpvl
Name: targeted mutation 1.1, Grace Pavlath
MGI accession ID: MGI:5888723
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence
Marker symbol: Pabpn1
Marker name: poly(A) binding protein, nuclear 1
Chromosome: 14
Strain of origin: B6.Cg-Thy1^a
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have the floxed sequence removed, allowing for expression of Pabpn1^A17 in cre-expressing tissues.
Molecular note: The gene was targeted with a construct containing the following elements: a loxP site, the wildtype cDNA, an internal ribosomal entry site (IRES), an enhanced green fluorescent protein (eGFP) reporter gene, an FRT site flanked neomycin resistance gene, a second loxP site, and the portion of the human cDNA representing a (GCG)7 alanine codon expansion. This was targeted to immediately downstream of the ATG start codon in exon 1 and adds 7 alanines to the endogenous 10. Subsequent Flp-mediated recombination removed the neo cassette. This alanine expansion allele mimics a mutation found in oculopharyngeal muscular dystrophy (OPMD) patients.
General note: Generated by Ozgene