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

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Genomics and Epigenomics

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

• PhITSeq screens performed with CDTs secreted by different

bacteria

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Results (cont.)

• Gene-trap insertions identified in loci essential for CDT

intoxication

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Results (cont.)

• Loci linked to 12 separate phenotypes

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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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The end

• Questions?

• Thanks!

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