This study aims to construct an inducible RNA interference system to knockdown the H1a and H1b genes in mouse embryonic stem cells. The system uses a vector construct containing shRNA cassettes against H1a and H1b, as well as expression vectors for a receptor that dimerizes in response to the drug ecdysone. This allows induction of RNA interference upon addition of ecdysone. The vector also contains antibiotic resistance and homologous arms for integration into the genome. Upon transfection into cells, the system will allow analysis of effects of H1 knockdown on cell morphology, cell cycle, and chromatin structure. This could provide insight into how chromatin structure influences cellular processes and identify new therapeutic targets.

1. Methods
Construction of inducible RNA interference system for H1 knockdown
Mahveesh Chowdhury, Yuhong Fan, Chenyi Pan
School of Biology, Parker H. Petit Institute of Bioengineering and Biotechnology, Georgia Institute of
Technology
Background
Vector Construct Plan
5’ and 3’ HPRT homology - allow homologous recombination of
vector into genomic DNA of mouse.
H1a and H1b shRNA cassette – required for H1a & H1b
knockdown
RXR and VgEcR – expression vectors that dimerize together to act
as receptor for ecdysone
Basticidin resistance gene – promotes antibiotic-resistance, to
select for positive clones containing vector construct.
Funding Source
National Institute of Health.
Georgia Institute of Technology, School of Biology
Potential Significance
Results
Uninduced Induced (5μM PonA)
Fluor Vis Fluor Vis
Figure 3. Fluorescence microscopy of transfected
uninduced ES cells (left), and ES cells induced with 5μM
Pon A (right) 55h after Fluor and Vis.
Figure 4. Western blot of H1a and H1b knockdown vector
pABIND in transfected HEK293 cells.
The samples in each lane consist of various combinations
of plasmids with no transfection (1), Flag-H1a expression
vector(2), Flag-H1b expression vector(3), Flag - H1a
expression vector + pEMS-VgRXR + pABIND(4), Flag -
H1a + pEMS-VgRXR + pABIND + PonA(5), Flag - H1b +
pEMS-VgRXR + pABIND(6), Flag-H1b + pEMS-VgRXR +
pABIND + PonA(7)
Objectives
Construction of a vector to knockdown H1a and H1b genes
using an ecdysone-inducible RNA interference system.
Investigate functions of H1 in:
Cell cycle
Gene expression
Chromatin structure
Summary
Approach
Utilization of RNA interference for knockdown of gene
expression has become a standard tool for the study of
gene function.
Linker histones are essential for chromatin folding and DNA
packaging in most eukaryotic cells. Recently, it has been
anticipated that H1 can influence gene expression as well.
This research examines the effects of silencing H1 genes
by using an inducible RNA interference system constructed
in vitro, and analyzing the effects on cell morphology, cell
cycle patterns, and chromatin folding. The vector construct
responds to drug analog ecdysone, which stimulates the
RNA interference system.
Figure 1. Schematic presentation of the two vector constructs Studying the effects of knockdown of H1 expression on
cellular phenotypes can provide critical clues to
understanding the relationship between high order
chromatin structure and folding, and various cellular
processes, which may lead to new targets for therapeutic
intervention.
α Flag
α β-actin
1 2 3 4 5 6 7
Relative intensity 5.0 47.7 1.1 0.5 20.1 2.9
Selection for zeocin antibiotic resistant clones
Western blotting to check knockdown
Chromosomal DNA staining
Coulter Counter for cell number counting (growth curve)
Culture positive clones
S
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B
C
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N
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Figure 2. Schematic presentation of experimental procedure including
subcloning, transfection and phenotype analysis done on mouse ES cells.
pABIND
4.3kb20 kb