Group 4 members:

gooseliverΒιοτεχνολογία

22 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

105 εμφανίσεις

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

2013/10/22

Group 4

1

Genomics and Epigenomics

Outline


A powerful forward genetic biotechnology for
phenotype related genes identification, genome
annotation……


Backgrounds


Biotechnologies


Results


Discussions


2013/10/22

Wang Ting

2

(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)

2013/10/22

Wang Ting

3


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.

2013/10/22

Li Jun

4

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
.

2013/10/22

Group 4

5

From google picture

Gene
-
trap insertion mutagenesis

2013/10/22

Li Jun

6

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;

2013/10/22

Li Jun

7

PhITSeq

2013/10/22

Jiang Bai

8


Processing


Insertional mutagenesis
-
> Phenotypic selection
-
> sequencing
-
>
Bowtie mapping to get insertion sites


Sequencing for selected population

2013/10/22

Jiang Bai

9

Short DNA sequences flanking the
inserted gene
-
trap vectors were amplified.


Results


PhITSeq screens performed with CDTs secreted by different
bacteria

2013/10/22

Qin Zhiyi

10

Results
(cont.)


Gene
-
trap insertions identified in loci essential for CDT
intoxication

2013/10/22

Qin Zhiyi

11

Results
(cont.)


Loci linked to 12 separate phenotypes

2013/10/22

Qin Zhiyi

12

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
a
nnotation, or comparative analyses;

2013/10/22

Wang Ting

13

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;


2013/10/22

Wang Ting

14

The end


Questions?


Thanks!

2013/10/22

Group 4

15