Experimental Protocols

• Component 1:
  • Genetrap overview (Sanger Center)
  • Blastocyst injections and mouse breeding
  • Growing feeder-independent embryonic stem cells
  • Electroporation and isolation of ES cell lines
  • PLAP staining protocol
  • (Taqman 7700 QPCR Assay) to assess neo dosage in knockout mice
  • Genotyping by dot blot analysis
  • 5'RACE sequence tag generation
  • 5'RACE sequencing outline
  • 5'RACE sequencing details
  • Genotyping mice
• Component 2:
  • Automated data processing of 5'RACE sequence tags
  • Trace file naming conventions
  • Vector splicing events and vector removal
  • Automated annotation of 5'RACE sequence tags
  • Linking in situ images to identified genes
  • Finding clones for identified genes
  • Manual identification protocol
• Component 3:
  • Paraffin embedding and sectioning of mouse embryos
  • In situ hybridization with DIG-probes
• Component 4:
  • Mouse heart harvest for DNA arrays
  • Tail tip digest and PCR protocol
  • X-gal staining protocol
  • Doxycycline administration protocol for mice
  • Arraying Protocols
    • Using ArrayMaker 2.1 Software with the Linear Servo Arrayer
    • Probe Synthesis and Fluorescent Labeling
    • Affymetrix GeneChip - expression analysis protocol
    • Microarray Hybridization
    • Preparation of poly-L-lysine coated slides
• Component 5:
  • B-gal staining protocol
  • B-gal expression in Acly
  • B-gal expression in Agpat6
  • B-gal expression in Man1
  • B-gal expression in Nmt1
  • B-gal expression in Scgn10
  • B-gal expression in Srebfp2
  • B-gal expression in Agpat4
  • B-gal expression in Fatty Acid Binding Protein 3
• Component 6:
  • Antigen (OVA) induced asthmatic animal model
  • Bleomycin induced pulmonary fibrosis
• Component 7:
  • Lung Organ Culture