Skip to main content

Table 1 PCR primers used for targeting vector construction, probe preparation, and mouse genotyping

From: Mutation-induced loss of APP function causes GABAergic depletion in recessive familial Alzheimer’s disease: analysis of Osaka mutation-knockin mice

Name

Sequence

Targeting vector

 

 TV-Am-5'-F (NotI)

5'-ATAAGAATGCGGCCGCGTAGGAAGGCCCAGCTAGAAGGAAATGGG-3'

 TV-Am-5'-R (NarI)

5'-CCGATGATGGCGCCTTTGTTCGAACCCACATC (ΔTTC) AGCAAAGAACACCTTCGAAAGGAAGCCG-3'

 TV-Am-M-F (NarI)

5'-CGGCTTCCTTTCGAAGGTGTTCTTTGCT-3'

 TV-Am-M-R (AscI)

5'-TTGGCGCGCCAGTTAACTAGGCCTAATGTTCCTCCATGGTAACCACGC-3'

 TV-Am-3'-F (PmeI)

5'-AGCTTTGTTTAAACAGGCTGTTGCCCTGAACTTCCACCTGAG-3'

 TV-Am-3' R (AatII)

5'-GGGGTTAGACGTCCCATTGGGTGTGACCCCACTTCAGAG-3'

Southern probes

 

 Am-5'-probe F

5'-TCCCCCACCCCCTGTTATAAAAGG −3'

 Am-5' probe R

5'-TGCTCTTTAAATCACCCCGGTTGC-3'

 Am-3'-probe F

5'-TCCTCTCGTCTTCCAACGCGGCTT −3'

 Am-3'-probe R

5'-CCGCCAGGCCAGAGCTCTACAGCA-3'

Genotyping

 

 KI/WT forward

5'-CCTAGGGACCCACCAACTCACGCT-3'

 WT reverse

5'-GGTGGAAGTTCAGGGCAACAGCCT-3'

 KI reverse

5'- TCTCCTGTCATCTCACCTTGCT-3'

  1. The targeting vector was constructed with three DNA fragments (5', middle, and 3') derived from mouse APP gene. The three fragments were produced by PCR using the primer pairs of TV-Am-5'-F/R, TV-Am-M-F/R, and TV-Am-3'-F/R, respectively. Underlines indicate restriction sites. ΔTTC represents a deletion of codon693 (GAA). Southern blot hybridization was carried out with 5' and 3' probes that were prepared by PCR using the primer pairs of Am-5' probe F/R and Am-3' probe F/R, respectively. Genotyping of mice was performed by PCR using the three primer mixtures. The KI reverse primer was in the neomycin-resistance gene. Wild-type allele produced a 780 bp PCR product with the primer pair of KI/WT forward and WT reverse, while KI allele showed an 1820 bp PCR product with the primer pair of KI/WT forward and KI reverse
Back to article page

Acta Neuropathologica Communications

ISSN: 2051-5960

Contact us