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Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun

Genomics and Epigenomics. Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun. Outline. A powerful forward genetic biotechnology for phenotype related genes identification, genome annotation…… Backgrounds Biotechnologies Results Discussions. (1). (2). Background.

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Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun

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  1. Genomics and Epigenomics Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun Group 4

  2. Outline • A powerful forward genetic biotechnology for phenotype related genes identification, genome annotation…… • Backgrounds • Biotechnologies • Results • Discussions (1) (2) Wang Ting

  3. Background • The ability to remove or inactivate single genes in cells is revolutionary; • Insertion mutagenesis in a haploid background can disrupt gene function, using retroviral gene-trap vector to generate insertions (Jan E. Carette et al. Science. 2009) • Extend by applying phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing Wang Ting

  4. Backgrounds (Authors intro.) • Jan E Carette: • A postdoc in the Brummelkamp lab • Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. • Papers: • Haploid genetic screens in human cells identify host factors used by pathogens. Science, November 27, 2009. • Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature, online on August 24, 2011. Li Jun

  5. Retrovirus • A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. • The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA. • Retroviruses are enveloped viruses that belong to the viral family Retroviridae. From google picture Group 4

  6. Gene-trap insertion mutagenesis International Gene Trap Consortium (IGTC) http://www.genetrap.org/tutorials/overview.html Li Jun

  7. Phenotype selection • CDTs for phenotype selection • Identify host factors required for the effects of backterial toxins; • to determine whether CDTs of diverse origin and structure use some common or different factors for their entry and intoxication; Li Jun

  8. PhITSeq • Processing • Insertional mutagenesis -> Phenotypic selection -> sequencing -> Bowtie mapping to get insertion sites Jiang Bai

  9. Sequencing for selected population Short DNA sequences flanking the inserted gene-trap vectors were amplified. Jiang Bai

  10. Results • PhITSeq screens performed with CDTs secreted by different bacteria Qin Zhiyi

  11. Results (cont.) • Gene-trap insertions identified in loci essential for CDT intoxication Qin Zhiyi

  12. Results (cont.) • Loci linked to 12 separate phenotypes Qin Zhiyi

  13. Discussions • advantages • Haploid cell line  powerful global gene disruption; • High throughput deep sequencing  analyze pools of cells, get genome-wide overviews of genes and enable rapid assessment of the spectrum of genes, assigning genes to phenotypes with high saturation and accuracy; • many phenotypes are accessible  efficient for the genome annotation, or comparative analyses; Wang Ting

  14. Discussions (cont.) • disadvantages • Rely on the use of one particular human near-haploid cancer cell line (gene function is condition-specific); • compared to RNAi-based screens (can be applied to many cell types, but cannot achieve global gene disruption); • Genetic redundancy or interaction among mutant alleles may affect the selection and statistical results; Wang Ting

  15. The end • Questions? • Thanks! Group 4

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