Expression Analysis of WD-Repeat Genes: Myelination Onset Study

1. Expression analysis of two novel WD-repeat genes during onset of myelination Samuel Adams July 13, 2006

2. Project Goal: To determine WHEN the gene is activated during development and WHERE the activation takes place.

3. DNA Definition of a gene A gene is a specific sequence of DNA carrying the information necessary for the production of a protein RNA Protein • Protein responsibilities: • Directed growth and development • Cell signaling • Breakdown of nutrients (enzymatic)

4. Gene Expression

5. How do we study genes? In mutant fish, certain genes have been disrupted, usually resulting in a protein that does not work properly or the complete absence of the protein. By looking at what goes wrong during the development of these mutants, we can rationalize what role the gene plays. Bad gene Good gene

6. Myelination

7. Adam Amsterdam, while working at MIT, developed a method of insertional mutagenesis to harvest ~315 different mutations in the genes of zebrafish. Also provided were pictures for a preliminary screening Megan West, a 2006 graduate of BW, determined which of the mutants would be beneficial to study defects in myelination, by staining for myelin and screening the embryos for defects. Identifying the Mutants

8. Picking the Mutants 2246 Day 3: slightly smaller head and eyes Day 5: small head and eyes, underdeveloped liver/gut, pericardial edema

9. Picking the Mutants 3630a Day 3: a bit smaller head and eyes Day 5: small head & eyes underdeveloped liver/gut

10. What do we do with mutants? 2246 After deciding which mutants to use, I went online to a site called NCBI to find the gene sequences. This site has almost the entire zebrafish genome sequenced. GAGGGGAGTTCACAGCAACAAGATGAGAGAGGAAGCCTTGAGATGGAGGCAGGGGGGTGATTTATCGTAA AAAATAGCGAATTTGGAGACAGTTGACATTCCGTTTTCAGAAAGAGATATAAAAGTCTCCCCAAGGAAGA GTCGTGTGTTAAATTTAGCTAACGTTATATTAATACTCAGTCAGCACTGGATATTTGGGTGAGTTCCTCA AGTATTGACCGGGCGCCATTTAAACACGCTGTGCGGGAGACAGGAAAGAAATAACCGCTCTCACAGCCCG CACTCGGCTTTCTGCTTCCGCGGACAGATTCTGCTCTCTAATAAACACACTTATTTGTTAGGAGATTGTA TTCTCAGTGTGGTAGGCAGTAGAAATGTGGCCTGTGTGTGAAGGCCTCTGGTAGCGCCAGGTATTGTGTG TGTTAGCTGGCTAACGGCTAACAGACCTCGAACTGAAGAGAGTTACGACCCCGAGCAAGAGACTACAGGT GTACTAAACCGTGACCATGGCGACTGACATCGGCTCCCCTCCGCGGTTCTTCCATATGCCTCGCTTCCAG CACCAGGCTCCCCGTCAGCTGTTCTACAAGAGGCCCGACTATGACCAGCAGCAGGCCATGCAGCAGCTCA CCTTTGACGGCAAGCGCATGAGAAAAGCCGTCAACCGCAAAACCATCGATTACAATCCATCCGTCATCAG ACATCTAGAGAATCGATTGTGGCAGCGAGATCATCGAGATTTCCGTGCTGTTCAACCAGACGCAGGCTGC TATAATGAACTCGTTCCCCCAGTGGGCGTGATTAGCAATCCTATGAATGCTGTCACGACAAAGTTTGTCA GAACCTCCACCAACAAAGTCAAATGCCCTGTGTTTGTTGTGAGGTGGACTCCTGAAGGCAGGCGACTTGT AACAGGTGCCTCCAGTGGGGAGTTTACTTTATGGAATGGGCTCACGTTCAACTTTGAGACAATTTTACAG GCTCATGACAGCCTTGTCAGGGCAATGACCTGGTCTCATAATTACATGTGGATGTTAACTGCCGACCATG GAGGTTATGTGAAGTACTGGCAGTCCAACATGAATAACGTCAAGATGTTCCAGGCTCACAAGGAGGCGAT TAGAGAGGCCAGTTTCTCTCCTACGGATAATAAATTTGCCACTTGCTCTGATGACGGAACTGTTCGCATC TGGGACTTTCTGCGCTGTCACGAGGAAAGAATCCTGAGAGGTCATGGAGCAGATGTGAAGTGTGTGGACT GGCATCCCACAAAGGGCTTGGTGGTGTCCGGCAGCAAAGACAGCCAGCAGCCCATCAAATTTTGGGACCC AAAGACTGGACAAAGCTTGGCAACACTACATGCACATAAAAACACAGTCATGGAGGTGAAGTGGAACCTC AATGGCAACTGGCTATTGACAGCATCACGAGACCATTTATGCAAACTGTTTGACATCCGCAACCTCAAAG AGGAGCTGCAGGTTTTTAGAGGACACAAGAAAGAAGCCACAGCTGTGGCCTGGCACCCAGTTCACGAAGG CATGTTTGCCAGTGGAGGTTCTGATGGATCACTACTGTTTTGGCATGTTGGTGTGGAGAAAGAGGTTGGA GGAATGGAGATGGCACATGAAGGAATGATCTGGAGTTTAGCCTGGCATCCGCTGGGCCACATTTTGTGTT CTGGGTCTAACGATCACACAAGCAAATTTTGGACGAGGAATCGGCCTGGGGACAAGATGCGGGACAGATA CAATCTAAATTTGTTGCCTGGAATGTCAGAAGATGGGGTGGAGTATGATGACATGGACACCAATAGTATT GCAGCCATTCCTGGCATGGGGATCCCAGAACAACTGAAGGCAGCCATGGAACAAGAACAATCAGGGAAAG ACATGGTGGCAGAGCCTGAGATGAGCATCCCGGGGCTGGACTGGGGCATGGAAGAGGTCATGCTGAGGGA CCAAAAAAAGGCCCCGACTAAAAAAGTTCCTTATGCAAAACCCATTCCAGCTCAGTTCCAACAGGCCTGG GCAGAGAATAAGGTTCCGGTGATGCCTGCTGGAGAAGTTCCAAAGGAAAGGAAGGATGAGAAGAAAGTAG ATAGCAAGAAGAAGACGCAAGCAGAGATTGAGCAGGAAATGGCTGCCCTGCAGTATACAAACCCAATGCT GCTCGAG

11. WD-repeat proteins Department of Biochemistry & Molecular Biology, 108 Althouse Lab, University Park, PA 16802

12. Next Step: RNA Isolation To determine WHEN the gene is expressed, RNA was isolated from 24- 48- and 72- hours post-fertilization embryos as well as 5 days post-fertilization. Performing Reverse Transcriptase reactions on these products results in DNA necessary for the next step...

13. Polymerase Chain Reaction 94° C 55° C Thermocycler machine cycles through the needed temperatures 30 times 72° C

14. These WD40 Proteins are expressed during early development. 2246 3630a 1000 1000 850 850 24 hpf 72 hpf 24 hpf 72 hpf DNA Ladder DNA Ladder Adult Brain Adult Brain 48 hpf 5 dpf 48 hpf 5 dpf

15. To find out WHERE: The same PCR products used to determine WHEN the gene is expressed are used in the first step of determining WHERE the genes are expressed. The PCR products were incorporated into a vector that was then transformed into competent E. coli.

16. Bacteria Cultures E. Coli cells were used to create clones of the genes. The clones can then be used as probes for the In-situ hybridizations.

17. Colony PCR After choosing 3 white or light blue colonies from the bacteria cultures, PCR’s were also used to analyze which of the colonies were transformed DNA Ladder 3630a 2246

18. Isolating the Plasmid After determining which colonies were transformed, the plasmids from the bacteria were isolated to be used as the probes for the upcoming In-situ hybridizations 3630a 2246 DNA Ladder

19. Linearizing the Plasmid

20. In-situ Hybridization • Allows for the expression of genes to be studied with little or high transcription amounts. • Use of non-radioactively labeled probes allows for high resolution and short developing time. The use of a DNA or RNA to detect mRNA in a cell.

21. In-situ Hybridization Whole Mount Zebrafish

22. Questions?