Journal: PLoS ONE

Article Title: Novel GATA6 Mutations in Patients with Pancreatic Agenesis and Congenital Heart Malformations

doi: 10.1371/journal.pone.0118449

Figure Lengend Snippet: A) DNA sequence of proband and parents showing a single base pair change from G in the parents to a T in the proband at nucleotide position c.1428+1 G>T. B) Diagram of the GATA6 protein containing a GATA family homeodomain and two DNA binding zinc fingers. Arrows indicate locations of GATA6 variants discovered in proband 1 and 2.

Article Snippet: The variant identified in proband 2 was generated in vitro using a wild-type GATA6 expression plasmid (GATA6 myc-DDK tagged ORF clone; Origene), and Stratagene site-directed mutagenesis (Agilent Technologies).

Techniques: Sequencing, Binding Assay, Zinc-Fingers

Journal: PLoS ONE

Article Title: Novel GATA6 Mutations in Patients with Pancreatic Agenesis and Congenital Heart Malformations

doi: 10.1371/journal.pone.0118449

Figure Lengend Snippet: DNA Sanger sequencing of proband 2 demonstrates he has a normal allele (upper) and a mutant allele (lower). The wild-type GATA6 sequence c964_970 (underlined region) is deleted in the mutant allele (arrow).

Article Snippet: The variant identified in proband 2 was generated in vitro using a wild-type GATA6 expression plasmid (GATA6 myc-DDK tagged ORF clone; Origene), and Stratagene site-directed mutagenesis (Agilent Technologies).

Techniques: Sequencing, Mutagenesis

Journal: PLoS ONE

Article Title: Novel GATA6 Mutations in Patients with Pancreatic Agenesis and Congenital Heart Malformations

doi: 10.1371/journal.pone.0118449

Figure Lengend Snippet: A) Human wild-type GATA6 and/or GATA6 Y323fsX21 labeled ‘GATA6 variant’ were co-expressed with Hnf4α promoter in INS-1 cells. Hnf4α promoter luciferase activity was measured and levels normalized to vector alone. *P = 0.00001. B) Western blot analysis of human GATA6 protein (W lane) and GATA6 Y323fsX21 (M lane) expressed in HEK 293T cells. Vector (V lane) only was a negative control and to approximate the size a protein ladder (L lane) was utilized. GATA6 was detected with a GATA6 antibody against the N-terminal of the protein and beta actin was measured as a loading control.

Article Snippet: The variant identified in proband 2 was generated in vitro using a wild-type GATA6 expression plasmid (GATA6 myc-DDK tagged ORF clone; Origene), and Stratagene site-directed mutagenesis (Agilent Technologies).

Techniques: Labeling, Variant Assay, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Negative Control

Journal: Nature protocols

Article Title: Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells

doi: 10.1038/nprot.2013.049

Figure Lengend Snippet: Troubleshooting table.

Article Snippet: Gelatin, 0.1% (wt/vol; Sigma-Aldrich, cat. no. G1890) made up in sterile dH 2 O (Sigma-Aldrich, cat. no. W1503) and autoclaved to dissolve PBS, 1×, sterile (Ca 2+ and Mg 2+ free; Gibco, cat. no. 14190-094) Trypsin-EDTA, 0.05% (wt/vol; Gibco, cat. no. 25300054) Xfect (Clontech, cat. no. 631320) or Lipofectamine 2000 (Invitrogen, cat. no. 11668030) Opti-MEM I (Invitrogen, cat. no. 51985) Bio-Rad Gene Pulser II Xcell eukaryotic system (Bio-Rad, cat. no. 377267) Cuvette, 4 mm (Cell Projects, cat. no. EP-104) Agarose (Sigma-Aldrich, cat. no. A9414) Maxi-prep kit (Qiagen, cat. no. 12262) Gata6 cDNA vector (Origene, cat. no. MC219384) Isopropanol (Sigma-Aldrich, cat. no. I9516) Sodium acetate (Fluka, cat. no. 71196) Wizard SV genomic DNA purification system (Promega, cat. no. A2360)

Techniques: Expressing, Plasmid Preparation, Transfection, Incubation