Integrated Genomics
(BSC 442)

Course Outline for Spring 2007

Growth, Manipulation, and Basic Genetics of  C. elegans
- transferring worms
- sexing males and hermaphrodites
- identification of phenotypes
- genetic crosses: unknowns and known

Propagation and Identification of Transgenic C. elegans and Isolation of Nematode Genomic DNA
(with a dominantly-marked gene fusion to lacZ)
- grow a population of worms in preparation for staining
- C. elegans genomic DNA isolation (for use later)

Expression Analysis of Transgenic C. elegans
- lacZ staining & microscopy to identify expression pattern
   - use of digital imaging for photo documentation

Transformation of S. cerevisiae with Two-Hybrid "Bait" Vector
(consisting of C. elegans cDNA for a given transgene fused to a DNA-binding domain)

Screen Yeast Two-Hybrid Library of C. elegans cDNAs for interacting genes
- involves transformation of yeast with library
- auxotrophic growth assays and lacZ filter assays for determining interactions
- tests to eliminate false positives

Isolation of library plasmids from "positive" yeast clones
- yeast DNA isolation and electroporation of E. coli
- mini-preps of E. coli DNA containing worm library vector

DNA sequencing (1 short run) of multiple library clones:
- use of sequence data to compare to C. elegans genome
  data in identifying putative interacting clones by genomic database searching

PCR
(to amplify upstream regulatory region of gene from C. elegans genomic DNA)
    - use of databases to construct PCR primers for ligation of amplified DNA

 Recombinant DNA and RNA interference (RNAi)

-subcloning of PCR-amplified fragment for generation of plasmid vectors for use in RNAi feeding methodology
-use of RNAi for gene activity knockdown to attempt to determine phenotypes
related to the genes identified in the protein interaction screen

Phenotypic Analysis and Bioinformatic Comparison of RNAi-treated C. elegans lines 

AND

Transformation of C. elegans
by Microinjection and Particle Bombardment (Demos)

COMPLEMENTARY BIOINFORMATICS COMPONENT OF COURSE

The following databases and internet sites are used in conjuction with specific student assignments to demonstrate and learn some of the currently available bioinformatics programs utilized in modern functional genomic analysis