STOCK Myl1^tm1(cre)Sjb Atg7^tm1Tchi Gaa^tm1Rabn/PuroMmjax

Stock number: 037139
Product name:

MLCcre:Atg7^F/F:GAA^-/-

Research areas: Cell Biology Research, Metabolism Research, Mouse/Human Gene Homologs

Description

MLCcre:Atg7^F/F:GAA^-/- mice a carry a skeletal muscle-specific cre recombinase (Myl1^tm1(cre)Sjb), a conditional Atg7 (autophagy related 7) allele, and a knockout of Gaa (glucosidase, alpha, acid). These mice may be used as a model of Pompe disease (a lysosomal storage disorder) in which an autophagy gene (Atg7) is inactivated in muscle. Suppression of autophagy in skeletal muscle in GAA KO mice lowers the accumulation of glycogen by 50-60%. This strain may be useful in studies of autophagy and therapeutic management of Pompe disease.

Detailed description

Pompe disease is an inherited disorder caused by a deficiency of alpha glucosidase, encoded by the GAA gene. Lysosomal glycogen accumulates in tissues, including muscles (most notably skeletal and cardiac) and the central nervous system resulting in cardiac and skeletal muscle myopathy. Enzyme replacement therapy (ERT) using recombinant human GAA is used to treat Pompe disease. ERT successfully clears lysosomal glycogen from the heart but is less effective in skeletal muscle due to autophagic debris.  This mouse model of Pompe disease (MLCcre:Atg7^F/F:GAA^-/-) combines the GAA knockout allele with a muscle-specific cre recombinase (Myl1^tm1(cre)Sjb, myosin, light polypeptide 1), and an Atg7 (autophagy related 7) allele with loxP sites flanking the cysteine active site. The presence of cre in the strain inactivates Atg7 in fast muscle fibers. MLCcre:Atg7^F/F:GAA^-/- mice develop the clinical signs of Pompe disease - muscle weakness, kyphosis, and muscle wasting (by 7-9 months of age). In addition, glycogen levels are reduced by 50-60% in fast muscle (but not cardiac or slow muscle), autophagy is suppressed in fast (gastrocnemius) muscle, and to a lesser degree in slow (soleus) muscle, and there are decreased numbers of lysosomes (and smaller in size) as compared to GAA^-/- mice. Treatment of MLCcre:Atg7^F/F:GAA^-/- mice with ERT increases muscle glycogen to normal levels.

Development

Dr. Rosa Puertollano (NIH) bred individual mutant mice to combine all three alleles creating the triple mutant MLCcre:Atg7^F/F:GAA^-/- mouse.

The Myl1^tm1(cre)Sjb was created in the laboratory of Dr. Steven Burden (New York University Medical Center). The targeting construct contains Cre recombinase with a nuclear localization signal (NLS), a loxP-flanked neomycin cassette inserted immediately upstream of the initiation ATG in exon 1 of the Myl1 (myosin, light polypeptide 1) gene on chromosome 1. The mutation was created by homologous recombination in (129X1/SvJ x 129S1/Sv)F1- Kitl⁺-derived R1 embryonic stem (ES) cells. PGK-neo was removed by crossing resultant mice with deleter CMV-cre animals. This strain was maintained on a mixed genetic background incorporating 129, C57BL/6 and FVB.

The Atg7^tm1Tchi allele was created in the laboratory of Dr. Tomaki Chiba (Tokyo Metropolitan Institute of Medical Science). The targeting construct contains a loxP site upstream of exon 14, exon 14 fused to a cDNA fragment encoding aa 1786-2097 (exons 15, 16, 17), a polyA signal sequence, a neomycin cassette, followed by a 2^nd loxP site, a splice acceptor site, and a cDNA fragment encoding aa 1669-1698 (exon 14 with a stop codon preceding the active site) of Atg7 (autophagy related 7) gene on chromosome 6. Exon 14 includes an active site cysteine residue essential for activation; it is disrupted by cre-mediated recombination. The mutation was created by homologous recombination in (C57BL/6NCrlj x CBA/JNCrlj)F1-derived TT2 embryonic stem (ES) cells .Immunoblot analysis indicates that the presence of the loxP sites and neo did not interfere with expression of Atg7. This strain was maintained on a mixed background.

The Gaa^tm1Rabn allele was created in the laboratory of Dr. Nina Raben (NIAMS, NIH). Exon 6 of the mouse Gaa (glucosidase, alpha, acid) gene on chromosome 11 was targeted with a termination codon and a neomycin cassette and flanked by loxP sites via homologous recombination in 129X1/SvJ-derived RW-4 embryonic stem (ES) cells to create this 6^neo (or PD) knock-out allele. Resulting chimeric males were bred with wild-type C57BL/6J females. This strain was maintained on a mixed C57BL/6J-129X1/SvJ genetic background.

 In 2022, males and females were sent to The Jackson Laboratory Repository. Upon arrival, embryos were cryopreserved.

Allele

Symbol: Myl1^tm1(cre)Sjb
Name: targeted mutation 1, Steven J Burden
MGI accession ID: MGI:1931135
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: mlc^cre; Mlc1f-cre; MLC-Cre; Mlcf-cre
Marker symbol: Myl1
Marker name: myosin, light polypeptide 1
Marker synonyms: CMYO14; CMYP14; Mcl3; MLC1; MLC-1; MLC1/3; MLC1F; Mlc3; MLC3f; MLC3-f; MLCf; MLC-f; Mylf; MYOFTA
Chromosome: 1
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Mice carrrying this allele express cre recombinase in skeletal muscle tissue, including gastrocnemius, tibialis anterior and biceps femoris muscles.
Molecular note: Insertion of an NLS-cre gene into exon 1, immediately upstream of the initiator ATG. An adjacent lox-P flanked neomycin cassette was removed by Cre mediated recombination in the final allele.

Allele

Symbol: Gaa^tm1Rabn
Name: targeted mutation 1, Nina Raben
MGI accession ID: MGI:2148550
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: 6^neo; Gaa^-
Marker symbol: Gaa
Marker name: glucosidase, alpha, acid
Marker synonyms: E430018M07Rik; IOPD; LOPD; LYAG
Chromosome: 11
Strain of origin: 129X1/SvJ
Molecular note: Insertion of a neomycin cassette into floxed exon 6. No transcript derived from this gene was detected in skeletal muscle of homozygous mice by RT-PCR analysis, and no encoded protein was detected by Western blot in liver extracts from homozygous mice.

Allele

Symbol: Atg7^tm1Tchi
Name: targeted mutation 1, Tomoki Chiba
MGI accession ID: MGI:3587769
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: Atg7^F; Atg7^Flox
Marker symbol: Atg7
Marker name: autophagy related 7
Marker synonyms: 1810013K23Rik; Apg7l; APG7-LIKE; GSA7; SCAR31
Chromosome: 6
Strain of origin: (C57BL/6NCrlj x CBA/JNCrlj)F1
Molecular note: A targeting construct was designed to insert a loxP site in intron of the 13th coding exon. Coding exon 14 was fused to a cDNA encoding coding exons 15, 16, and 17 together with a polyadenylation signal after the stop codon. This was then followed by a neomycin resistance cassette and a second loxP site. An additional exon 14 sequence with a stop codon preceding the active site cysteine codon was inserted after the second loxP site. Immunoblot showed that the presence of the loxP sites and neo did not interefere with translation.