This "humanized" App knock-in strain, in which 3 point mutations were introduced into exon 14 of the mouse App gene, produces a mutant protein that is expected to more readily aggregate. Exon 14 is also flanked by loxP sites. This strain may be useful for studying late-onset sporadic Alzheimer's disease.
Frank LaFerla, University of California, Irvine
hAbeta-loxP-KI knock-in is a mutant "humanized" App allele in which three mutations were introduced into the mouse App exon that encodes the Aβ fragment (see below), such that the sequence now encodes the same amino acids as human. This exon is also flanked by loxP sites. These hAbeta-loxP-KI mice express mouse APP protein with a "humanized" amyloid beta peptide sequence. Homozygotes are viable and fertile. As the mice are characterized, we will modify the strain description and add phenotype data.
Important note about App isoforms and exon numbering: The App-201 isoform (695 aa protein) is encoded by 16 exons, of which the Abeta sequence is encoded by exon 14. The App-206 isoform (770 aa protein) is encoded by 18 exons, of which the Abeta sequence is encoded by exon 16.
The hAbeta-loxP-KI allele is available on a C57BL/6J congenic background (Stock No. 031050), on a C57BL/6NJ congenic background (Stock No. 032013) and on a mixed C57BL/6J and C57BL/6NJ background (Stock No. 030898).
The mouse BAC RP23-424A20, containing the entire App gene, was used to amplify and subclone a 3.6kb KpnI-PacI region containing App exon 14 (ENSMUSE00000131684). The Abeta coding sequence within exon 14 was modified by changing three bases to produce three amino acid substitutions (Aβ-encoding amino acids 5 (G->R), 10 (F->Y) and 13 (R->H)). loxP sites were added to flank exon 14 and a FRT site flanked neo selection cassette (pM-30) was used to enable G418-selection in mouse ES cells. The targeting vector was electroporated into C57BL/6N-derived JM8.N4 ES cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric male animals were bred to Actin-FLPe transgenic female mice, N5 on C57BL/6NTac, (see for example, Stock No. 005703), to remove the neo selection cassette. Male offspring mice that no longer carried the selection cassette were then bred to C57BL/6J females to remove the Tg(ACTFLPe)9205Dym transgene. The mice were then intercrossed to produce homozygotes that were subsequently intercrossed for 7 to 8 generations to generate homozygotes. The mice were then bred to C57BL/6NTac wildtype mice once. Heterozygotes were intercrossed to generate homozygotes for the hAbeta-loxP-KI (APPKI-hAbwt) allele that are also homozygous for wildtype Nnt. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304). The mice were backcrossed to C57BL/6NJ (Stock No. 005304) using a marker assisted, speed congenic, procedure.
|Allele Name||targeted mutation 1.1, Frank LaFerla|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Humanized sequence)|
|Gene Symbol and Name||App, amyloid beta (A4) precursor protein|
|Strain of Origin||C57BL/6N|
|Molecular Note||The targeting construct includes a loxP site upstream of exon 14, 3 point mutations inserted into exon 14 to create amino acid substitutions (amino acids 5 (G->R), 10 (F->Y) and 13 (R->H)) (ENSMUSE00000131684), a loxP site downstream of exon 14 and a FRT-flanked neo selection cassette, which is removed by FLP-mediated recombination.|
When maintaining a live colony, these mice can be bred as homozygotes.
When using the hAbeta-loxP-KI on B6NJ mouse strain in a publication, please cite the originating article(s) and include JAX stock #032013 in your Materials and Methods section.