Mutagenesis and Cloning of LRP6 and Ppp1r11

Mutagenesis of LRP6^VA and LRP6^(VA)P(PA) was performed by PCR-amplifying full length LRP6 with Phusion^TM DNA polymerase using primers designed to mutate selected amino acids into alanines: LRP6^VA forward: 5’-GATTCAGAACCTGCGCCCCCACCTCCCACACCC-3’; LRP6^(VA)P(PA) forward: 5’-GATTCAGAACCTGCGCCCGCACCTCCCACACCC-3’; common reverse: 5’-GGGTGTGGGAGGTGCGGGCGCAGGTTCTGAATC-3’. GoTaq (Promega; Cat# M7841) DNA polymerase was used to amplify full-length Ppp1r11 using cDNA obtained from whole X. tropicalis embryos (Forward: 5’-TAAGCAGAATTCATGGCAGAATCCTCCGGG-3’; Reverse: 5’-TGCTTACTCGAGTTAGTGTTGCATGCTGCC-3’). Where indicated, N-terminal Flag tags were added via forward primers (Forward: 5’-TAAGCAGAATTCATGGATTACAAGGATGACGACGATAAGGCAGAATCCTCCGGGCCG-3’). The GSK3 ciliary biosensor (pArl13b-GSK3) was generated as follows: mouse Arl13b CDS without stop codon and the coding sequence of a linker peptide (Glycine-Glycine-Glycine-Glycine-Serine) were inserted upstream of the GFP coding sequence in a pCS2-GFP-GSK3-MAPK-Flag backbone plasmid (kind gift from De Robertis lab, see ref. ^{39 (link)}). The mArl13b-linker part was amplified from E13.5 mouse whole brain cDNA by using the forward primer 5’-TTGCAGGATCCGCCACCATGTTCAGTCTGATGGCCAACTG-3’, and the reverse primer 5’-CTTGCTCACTGATCCTCCTCCTCCTGAGATCGTGTCCTGAGCAT-3’. The Linker-pCS2-GFP-GSK3-MAPK-Flag part was amplified from the pCS2-GFP-GSK3-MAPK-Flag backbone with the forward primer 5’-CGATCTCAGGAGGAGGAGGATCAGTGAGCAAGGGCGAGGAGCT-3’ and the reverse primer 5’-CAGTTGGCCATCAGACTGAACATGGTGGCGGATCCTGCAA-3’. Gibson assembly was performed according to the protocol from the NEB Gibson assembly kit (#E5510). Positive clones were picked and confirmed by Sanger-sequencing.

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Seidl C., Da Silva F., Zhang K., Wohlgemuth K., Omran H, & Niehrs C. (2023). Mucociliary Wnt signaling promotes cilia biogenesis and beating. Nature Communications, 14, 1259.

Publication 2023

Alanines Amino acids Backbone Biosensor Brain Cdna Clones Coding sequence Dna polymerase Embryos Glycine Gsk3 Lrp6 Mouse Mutagenesis Peptide Plasmid Primer Promega Serine Stop codon

Corresponding Organization :

Other organizations : DKFZ-ZMBH Alliance, Heidelberg University, University Hospital Münster

Top 5 similar protocols

Variable analysis

independent variables

Amino acid substitutions in LRP6 protein (LRP6^VA and LRP6^(VA)P(PA))
Addition of N-terminal Flag tags to Ppp1r11 protein

dependent variables

Unmeasured, likely related to the effects of the LRP6 mutations and Ppp1r11 tagging on cellular or molecular processes

control variables

Full-length LRP6 protein
Untagged Ppp1r11 protein

controls

Positive control: Full-length LRP6 protein
Negative control: Untagged Ppp1r11 protein

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