There were errors in Disease Models & Mechanisms (2022) 15, dmm049330 (doi:10.1242/dmm.049330).The authors recently discovered that one of the Cre-dependent transgenic mouse lines used in the study is not correctly described within the article. The mouse line labelled as R26-APP in the paper was originally described as containing both the Swedish and Iberian mutations, but carries only the Iberian mutation. This was discovered by The Jackson Laboratory when they sequenced through the complete insert to confirm the allele before entering the line into their repository for distribution. The authors also discovered an unintended point mutation located just outside the Aβ domain, M647T by APP695 numbering (M722T by APP770 numbering). They propose that the model should be identified as R26-APPIbe rather than as R26-APP.A diagram of the allele that appears in Fig. 2A has been corrected:In addition, a section describing the cloning methods in the Materials and Methods has been updated. The corrected and original versions are shown below:‘Cloning of R26-LSL-hAPPIbe transgene’ section (corrected). Human APPIbe 695 amino acid cDNA was generated in two steps. The N-terminal 1592 nucleotides of huAPP695 were cut from pcDNA3.1-sAPPα (a gift from Hui Zheng, BCM) with HindIII and SacI and subcloned into the same sites of transfer plasmid L4440 (Addgene, #1654). The remaining fragments were amplified from a human cDNA library using a two-step PCR method. Both PCR products contained the Ibe mutation as well as an overlap region needed to serve as an extended template for the second PCR. The first C-terminal fragment was amplified using hAPP C-terminus forward primer 5′-AACGAAAACCACCGTGGAGCT-3′ and Ibe reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′. The second C-terminal fragment was amplified by Ibe forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and hAPP C-terminus reverse primer 5′-GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG-3′. The complete hAPP695Ibe C-terminus was then amplified from a mixture of the two shorter products using hAPP CT forward primer and hAPP CT reverse primer. The purified huAPPIbe C-terminal PCR fragment was digested with EcoRV and SacI and ligated into the EcoRV/SacI sites of L4440-APP(1-1592), resulting in L4440-APPIbe. The complete huAPPIbe sequence was transferred into an intermediary expression plasmid, which was digested with MluI and FseI and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) to generate the final transgene construct pJJ-hAPPIbe. The modified Ai3 targeting vector retained the original Ai3 Cre-dependent CAG-loxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).‘Cloning of R26-LSL-hAPPSwe/Ibe transgene’ section (original). Human APPSwe/Ibe 695 amino acid cDNA was amplified from the pBR322-APPSwe/Ind plasmid used to generate the Tg CRND8 mouse model (Chishti et al., 2001) (a gift from David Westaway, University of Alberta, Canada) with primers that converted the original Indiana mutation V717F into the Iberian mutation I716F (point mutation numbering is for the standard human APP 770 amino acid isoform). The N-terminus of APPSwe/Ibe 695 was amplified using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′; the C-terminus was amplified using the forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The resulting hAPPSwe/Ibe fragments were combined by amplification using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The complete hAPPSwe/Ibe 695 cassette was digested with MluI and FseI, and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) that had been cut with the same enzymes to generate the final transgene construct pJJ-hAPPSwe/Ibe. The modified Ai3 targeting vector retained the original Cre-dependent CAGloxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).Throughout the paper, where the text refers to R26-APP, R26-APPSwe/Ibe or ROSA26-CAG-LSL-APPSwe/Ibe, corrections have been made to R26-APPIbe or ROSA26-CAG-LSL-APPIbe. The model will still be distributed by The Jackson Laboratory as stock #037319, but using the line name R26-APPIbe. The study results and conclusions remain unaffected.Both the online full-text and PDF versions have been updated. The authors apologise to readers for these errors and any inconvenience they may have caused.
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Jul 18, 2024
Haruka Munezane + 5
Open Access
