1. NGUYEN THI NHI - Master’s student
Department of Physiology
Keimyung University School of Medicine
Progress report
2. Finished works in 8th week
1. Practice
Media making, cell thawing, cell culture, cell passage
Tissue extraction
2. Theory
Main principle of Cre-loxP System, Chromatin immunoprecipitation
(ChIP) assays, and EdU Cell Proliferation Assay
4. Cre-loxP System
• To generate the spatiotemporally controlled mutant mice, two
elements are needed in the Cre-loxP system.
• First, Cre-driver strain is generated in which Cre recombinase is
expressed by a promoter that specifically targets the cell or
tissue of interest.
• Second, loxP flanked (floxed) DNA containing mouse strain is
needed to be generated.
Conditional knockout mice are then generated by breeding the
Cre-driver strain with a floxed mouse strain.
Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes
DOI: https://doi.org/10.5625/lar.2018.34.4.147
5. Mechanism of Cre-loxP system
(A) An overview of Cre-loxP system. 38 kDa Cre recombinase recognizes the loxP sites of
specific 34 bp DNA sequences.
(B) General breeding strategy for conditional mutation using loxP and Cre driving mouse line.
In principle, one mouse must have a tissue-specific driven cre gene and another mouse have
loxP flanked (floxed) alleles of interest gene Y. Expression of Cre recombinase excises floxed loci
and inactivates the gene Y.
7. • Protein and associated chromatin in living
cells or tissues are temporarily bonded. The
DNA–protein complexes are then sheared into
∼500 bp DNA fragments.
• Cross-linked DNA fragments associated with
the proteins are selectively
immunoprecipitated from using appropriate
protein-specific antibody.
• Then the associated DNA fragments are
purified and their sequence is determined.
The DNA undergoes PCR amplification using
primers targeting a particular genomic locus.
• These DNA sequences can be subjected to a
series of downstream analysis techniques.
Song, C., Zhang, S., and Huang, H. (2015). Choosing a suitable
method for the identification of replication origins in microbial
genomes. Front. Microbiol.
Principle
8. There are two general procedures for carrying out ChIP experiments:
native ChIP (N-ChIP) and cross-linking ChIP (X-ChIP)
N-ChIP X-ChIP
Native chromatin is used as the
substrate (proteins are not cross-
linked to the DNA).
Proteins are cross-linked to the DNA.
Fragmentation of the chromatin is
achieved by micrococcal nuclease
digestion, resulting in a nucleosome
based resolution.
Cross-linking is typically achieved
using formaldehyde and chromatin
is fragmented by sonication, creating
random fragments.
N-ChIP is restricted to proteins that
are very tightly associated with
chromatin, typically limiting this
type of ChIP to histones and their
modifications.
As the proteins are cross-linked to
the DNA a broad range of chromatin
associated factors can be analyzed
using this technique.
10. EdU Cell Proliferation Assay
Cells grown in the presence of 5-EdU incorporate the compound at
thymidine bases during S-phase. Fluorophore-labeled azide reacts with
the incorporated EdU to allow detection by microscopy or flow
cytometry.
11. EdU Cell Proliferation Assay process
Detection of actively proliferating
cells can be performed using
detection protocols for either image
based or flow cytometric analysis
12.
13. • Objective: to determine the role of insulin signal at β cell level.
• They created βIRKO mice by Cre-loxP mediated recombination
technique. They compared result between βIRKO mice and 3 controls
groups (IRlox, Rip-Cre, and WT).
- IRlox: to rule out the effect of introduction of loxP sites in the
insulin promoter
- Rip-Cre: mice carrying only the Cre transgene on a rat insulin, to
rule out any effect of Cre expression on β cell function
- WT
14. Result
They assessed the effect of β cell insulin receptor KO on glucose
homeostasis, glucose and insulin level in the fasted and random-fed
states.
• The fasting insulin levels in βIRKO mice increased compared with 3
other groups.
• βIRKO mice exhibited a decrease in acute insulin release response to
Glucose.
• After 30min of glucose challenge, insulin levels did gradually rise
suggest the retention of second phase insulin secretion in βIRKO
mice .
15. Result
• They assessed the impact of altered 1st phase insulin release on the
ability of glucose tolerance. (intraperitoneal injection)
βIRKO mice showed a significant higher glucose levels than
controls, and glucose tolerance continued worsen with age.
• They evaluated morphology of β cell by electron microscopy and
revealed well-preserved structure of cell with no apparent
differences in the cell membrane, endoplasmic reticulum, Golgi, …
• Immunohistochemical study showed a decrease (20-40%) in islet size
in 4-month-old βIRKO, but the distribution of GLUT2 remained
unchanged. Besides, the insulin content in this age was smaller than
controls.
16.
17. Insulin Regulates CENP-A and PLK1 Gene
Expression in Pancreatic β Cells
• The expression of CENP-A and PLK1 was significantly decreased in
primary β cells obtained from βIRKO mice compared to control.
• CENP-A protein’s peak expression was delayed 6-12 hr, and PLK1
was virtually undetectable throughout the cell cycle.
• Insulin binding to its receptor enhances CENP-A and PLK1
expression.
These results indicate the effect of glucose on CENP-A and PLK1
expression is dependent on the insulin receptor
18. Insulin-Stimulated FoxM1 Binding to CENP-A
and PLK1- Associated DNA in the Nucleus
• In mouse, FoxM1 was mostly localized to the nucleus in β cells. The
MAPK/ERK kinase (MEK)1/2 inhibitor (U0126) promoted nuclear
export of FoxM1.
• In human, FoxM1 was mainly localized to the cytosol in β cells of
cultured human islets after starvation. Insulin stimulation
facilitated nuclear localization of FoxM1.
(by immunocytochemistry and immunoblotting)
• FoxM1 was recruited to CENP-A and PLK1 genomic regions in
control β cells. And the binding was significantly reduced in βIRKO.
(using chromatin immunoprecipitation)
19. Insulin-Induced CENP-A Expression in
Human Islets Is Impaired in Type 2 Diabetes
• RT-PCR and qPCR analysis revealed significantly low levels of CENP-
A, and a trend to decreased expression of PLK1.
• The number of CENP-A expressing β cells was significantly
attenuated in the islets from T2DM donors. The expression of
CENP-A and PLK1 was increased by exogenous insulin treatment of
islets from non-DM donors.
Reduced CENP-A expression is decreased in islets from patients
with T2DM and it is associated with insulin stimulation.
20. β Cell-Specific CENP-A Knockout Mice
Demonstrate Reduced β Cell Proliferation
• β CenpaKO mice exhibited genomic recombination of Cenpa alleles and
consequent diminished expression of CENP-A only in islets and not in the
hypothalamus, liver, or spleen.
• The knockouts developed glucose intolerance, impaired insulin secretion,
decreased phospho-histone H3 (pHH3)+ mitotic and bromodeoxyuridine
(BrdU)+ proliferating β cells, and β cell mass by 24 weeks of age.
• CENP-A-deficient β cells from β CenpaKO mice demonstrated a
significant decrease in cell proliferation in response to a glucokinase
activator.
β CenpaKO mice exhibited a significant decrease in serum insulin levels,
β cell mass, mitosis, and proliferation compared with controls. CENP-A
plays an important role in the regulation of proliferation and the
maintenance of β cell mass.
21. Role of CENP-A and PLK1 in β Cell Proliferation
and Survival
• Knockdown of CENP-A reduced viability and metabolic activity in
control, IRS1KO, or IRS2KO β cells, but not in β IRKO cells. CENP-A
knockdown significantly increased G2/M-phase cells, suggesting
G2/M cell-cycle arrest.
• PLK1 knockdown reduced cell proliferation in control and β IRKO β
cells (evaluated by both the MTT and EdU incorporation assays).
• The expression of PLK1 was reduced in CENP-A knockdown cells,
while conversely CENP-A expression was diminished in PLK1
knockdown cells.
22. PLK1-Dependent CENP-A Deposition to
Centromere in β Cells
• CENP-A deposition at the mouse centromere marker was
attenuated in both FoxM1 inhibitor-treated control β cells and
intact βIRKO cells (in ChIP analysis).
• PLK1 knockdown and treatment with the PLK1-specific inhibitor (BI-
2536) repressed CENP-A binding at the centromere site.
• The process of CENP-A binding to the centromere was reduced in
human islets when using the IR and IGF1R dual inhibitor, the PI3K
inhibitor, or the PLK1 inhibitor.
23. Schematic of the regulation of CENP-A expression and its deposition to centromere in b cells.
