1. [Intermediate Plasmid 1]: Amyloid-β1−42 sequence was amplified by PCR from pET-SUMO-Abeta (Kirstein Lab) with primers cagtcaaccggtcagtcagctagcGATGCAGAATTCCGACATGAC and ataagaatctcgagctaCGCTATGACAACACCGCCCACC.
a. PCR-product and pPD95−77_EGFP (Fire Lab) were digested with AgeI and XhoI.
b. Amyloid-beta(1−42) sequence replaced EGFP sequence.
2. [Intermediate Plasmid 2]: wrmScarlet sequence was amplified by PCR from pSEM87_twk-18_mScarlet (Boulin Lab, 2017) with following primers: cagtcaaccggtcagtcatctagaATGGTCAGCAAGGGAGAGGC and ataagaatgctagcCTTGTAGAGCTCGTCCATTCC. Digestion was performed with AgeI and NheI to insert wrmScarlet upstream of Amyloid-beta(1−42).
3. [Intermediate Plasmid 3]: The rgef-1 promoter was amplified by PCR from pPD95-DBN(wt)-YFP (Kreis et al., 2019) with following primers: cagtcagcatgcCAAGACTAATTTTCGATTAACC and ataagaatatcgatataagaataccggtCGTCGTCGTCGTCGATGCCGTC. Digestion was performed with SphI and AgeI to insert rgef-1p upstream of wrmScarlet-Abeta. (IntermediatePlasmid_03)
4. [Intermediate Plasmid 4] Signalpeptide-Amyloid-β1−42 (SigPep-Abeta) was created by Fusion-PCR using a synthetic 50mer (gttttgctggcactgttctttatctttctggcaccagcaggtaccGACGCG) as first PCR template and CTATAGATCGATGCATAAGGTTTTGCTGGCACTGTTC plus CATGTCGGAATTCTGCATCCGCGTCGGTACCTGCTGG as primers; second PCR template was pET-SUMO-Abeta and CCAGCAGGTACCGACGCGGATGCAGAATTCCGACATG plus TCATCGACCGGTTTACGCTATGACAACACCGCC as primers.
a. The subsequent Fusion-PCR was performed with the products of the first and second PCR as templates and CTATAGATCGATGCATAAGGTTTTGCTGGCACTGTTC plus TCATCGACCGGTTTACGCTATGACAACACCGCC as primers.
b. Digestion was performed with ClaI and AgeI. The product was inserted into IntermediatePlasmid_03, as a result, the promoter sequence lost 400 base pairs. (IntermediatePlasmid_04)
5. [Intermediate Plasmid 5] hsp-3 IRES element was cloned from genomic DNA with the following primers: CCTATGACCGGTTGCTCTCCCTTCACCACTCC and GGATACTCTAGAGCCCAACAAGAATAAGGTCTTCATA.
Digestion was performed with AgeI and XbaI before insertion into Intermediate Plasmid 4 between SigPep-Abeta and wrmScarlet-Abeta.
6. The final Signalpeptide-Amyloid-β1−42-IRES sequence was amplified from Intermediate Plasmid 5 with the following primers: CTAACTCCCGGGATGCATAAGGTTTTGCTG and TGACTGCCCGGGTGAGCCCAACAAGAATAAGGTC.
Digestion was carried out with XmaI for insertion into IntermediatePlasmid_03 to create the final plasmid that contains all fragments. (pPD95_rgef-1p::SigPep-Aβ1−42-IRES-wrmScarlet-Aβ1−42::unc-54(3′UTR))
7. The neuronal wrmScarlet control was generated by cleaving pPD95.77 with SphI and EcoRI, removing GFP.
The promoter of rgef-1 was amplified with the primers cagtcagcatgcCAAGACTAATTTTCGATTAACC plus ataagaatatcgatataagaataccggtCGTCGTCGTCGTCGATGCCGTC and cleaved with SphI and NheI; wrmScarlet was amplified with the primers GATTAGCTAGCATGAAGACCTTATTCTTGTTGG plus GACTAGAATTCCTACTTGTAGAGCTCGTCCATTCC and digested with NheI and EcoRI. The plasmid was ligated with both fragments in one step.
8. The construct for muscular expression of Aβ1−42 with substoichiometric expression of wrmScarlet Aβ1−42 was generated by Gibson Assembly (NEB):
a. The promoter sequence of myo-3 was amplified from pCFJ104 with homologous overlaps to the construct for neuronal expression of Aβ1-42 (pPD95_rgef-1p::SigPep-Aβ1−42-IRES-wrmScarlet-Aβ1−42::unc-54(3′UTR): GCAAAACCTTATGCATCCCGGTCGTCATTTCTAGATGGATCTAGTGGTCGTG and CTTGGAAATGAAATAAGCTTGCATGAGTGATTATAGTCTCTGTTTTCGTTA.
b. The plasmid was amplified with primers bearing matching homologous overlaps, excluding the promoter of rgef-1: CACGACCACTAGATCCATCTAGAAATGACGACCGGGATGCATAAGGTTTTGC and TAACGAAAACAGAGACTATAATCACTCATGCAAGCTTATTTCATTTCCAAG.
9. The muscular wrmScarlet control was cloned by amplifying the promoter of myo-3 with the primers GCTGAACCGGTAGTGATTATAGTCTCTGTTTTCGTTAAT and GATTAGCTAGCCATTTCTAGATGGATCTAGTGGTCG.
a. The product was inserted into the neuronal control vector after digestion using AgeI and NheI, replacing the promoter of rgef-1.
10. The construct STOP-Aβ, for expressing Aβ1−42 only was generated by cleaving the neuronal Aβ1−42 expression construct with AgeI to remove SigPep-Aβ1−42.
a. The two oligonucleotides CCGGATGGGTGCATAAGCATAAC and GGCCGTTATGCTTATGCACCCAT were phosphorylated using T4-polynucleotide kinase and annealed by a gradient from 95 °C (5 min) to 25 °C (over 15 min).
b. The annealed construct was ligated into the AgeI cleavage site of the vector.
11. To achieve cell-specific knockdown of Aβ1−42, a short hairpin construct of the peptide was generated and expressed under the control of cell-specific promoter regions (Esposito et al., 2007).
12. The short hairpin construct against Aβ1−42 in URY neurons was generated by amplifying Aβ1-42 with the primers GCATTGCATGCGCTATGACAACACCGC and GCATTGCATGCGATGCAGAATTCCGACATGAC.
a. Both, amplificate and the plasmid pPD95.77 were digested with SphI to insert the PCR product and correct orientation of the insert was confirmed by sequencing.
b. Inverse Aβ1−42 was cloned with the primers GCATTGGATCCGCTATGACAACACCGCC and GCATTGCTAGCGATGCAGAATTCCGACATG; the splice leader 2 sequence from gdp-2 was amplified with the primers GCATTGCTAGCGCTGTCTCATCCTACTTTCAC and GCATTACCGGTGATGCGTTGAAGCAGTTTC.
c. Both were cleaved with BamHI, NheI and AgeI and inserted into the prepared vector pPD95.77 + Aβ1−42, which had been cleaved with BamHI and AgeI to generate pPD95.77 + Aβ1−42for/rev::sl2. Aβ1−42for/rev::sl2 was amplified with the primers GGATTGGTACCGCATGCGATGCAGAATTCCGAC and GCATTGGTACCGGTGATGCGTTGAAGCAG cleaved with KpnI for insertion into the vector pJB253 bearing tol-1p::GFP.
13. To express the short hairpin construct against Aβ1−42 in IL2 neurons of C. elegans, the promoter of klp-6 was amplified from pEY54 using the primers cagtcagcatgcGTTGGAAAGTTTGGTAAGTTGC and ataagaatggtaccatGGTATTCTGAAAAGTTCAAC.
a. Digestion was carried out with SphI and KpnI to insert the amplified fragment into pPD95.77.
b. Subsequently, the sequence for Aβ1−42for/rev::sl2 was amplified with the primers ataagaataccggtGATGCAGAATTCCGACATGAC and ataagaatctcgagGATGCGTTGAAGCAGTTTCC and inserted behind the promoter by restriction digestion with AgeI and XhoI, respectively, partially replacing the GFP sequence in pPD95.77.
14. Plasmid verified by sequencing