Pgem t easy vector

The full-length BN or FHH rat Shroom3 cDNA was amplified using PCR and cloned into a pGEM-T Easy vector (Promega) using the manufacturer’s protocol. BN-FHH recombinant Shroom3 plasmids were generated by cloning with restriction enzymes. The pGEM-T Easy vector containing the full-length BN or FHH Shroom3 cDNA was digested with two enzymes, AgeI and AgrAI, AgrAI and BstEII, or BstEII and NcoI (New England Biolabs). Desired plasmid fragments were gel-extracted and subsequently ligated using T4 ligase (New England Biolabs) to create Shroom3∆641–3044, Shroom3∆3044–4117, and Shroom3∆4117–5966 plasmids (number indicates the base pairs of cDNA replaced by FHH alleles). To create the single amino acid substitution mutant allele, the full-length BN Shroom3 cloned into the pGEM-T Easy vector was PCR-amplified using Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific) and phosphorylated primers harboring the mutated sequence. PCR products were subsequently isolated and ligated using T4 ligase. To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis. See Supplemental Table 6 for the sequence of PCR primers used in this study. All plasmids were sequenced and verified.

Genomic DNA isolated from white blood cells of disease-free individuals was obtained from the Van Andel Research Institute Pathology and Biorepository Core. For trio analysis, the following DNA samples were obtained from the National Human Genome Research Institute Sample Repository for Human Genetic Research at the Coriell Institute for Medical Research: HG00731, HG00732, HG00733, HG01938, HG01939, and HG01940. Bisulfite conversion and clean-up of 2 μg of genomic DNA was performed by using the EZ DNA Methylation Kit (Zymo Research) according to the manufacturer’s protocol. Locus-specific PCR was performed by using primers specific for bisulfite-converted DNA and PCR products cloned by using the pGEM-T Easy vector and NEB 5-alpha–competent Escherichia coli (New England Biolabs). Colonies were screened for positive inserts by PCR and sequencing performed using the M13 promoter.