Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun 22/3/27 Group 4 1 Genomics and Epigenomics
Dec 18, 2015
Outline
A powerful forward genetic biotechnology for
phenotype related genes identification, genome
annotation……
• Backgrounds
• Biotechnologies
• Results
• Discussions
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(1)
(2)
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)
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Extend by applying
phenotypic interrogation
via tag sequencing
(PhITSeq) to examine
millions of mutant alleles
through selection and
parallel sequencing
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.
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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.
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From google picture
Gene-trap insertion mutagenesis
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International Gene Trap Consortium (IGTC)
http://www.genetrap.org/tutorials/overview.html
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;
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PhITSeq
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• Processing
– Insertional mutagenesis -> Phenotypic selection -> sequencing ->
Bowtie mapping to get insertion sites
Sequencing for selected population
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Short DNA sequences flanking the inserted gene-trap vectors were amplified.
Results (cont.)
• Gene-trap insertions identified in loci essential for CDT
intoxication
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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;
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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;
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