the role of krüppel-like factor 2 in erythroid cells
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The Role of Krüppel-Like Factor 2 in Erythroid Cells. Bryan Castro Joyce Lloyd (Mentor ) Virginia Commonwealth University Dept of Human and Molecular Genetics. Background Information. Sickle Cell Anemia Hemoglobin KLF2 β-Globin Promoter Cre Recombinase. Sickle Cell Anemia. - PowerPoint PPT PresentationTRANSCRIPT
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Bryan Castro Joyce Lloyd (Mentor )
Virginia Commonwealth University Dept of Human and Molecular Genetics
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• Si ck le Cell Anemia
• Hemoglobin
• KLF2
• β-Globin Promoter
• Cre Recombinase
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Sickle Cell Anemia Hemoglobin that sickles Inherited Hemoglobinopathy
Predominant in people of African or Mediterranean ancestry
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Sickle Cell Anemia
Noguchi
Genetic mutation of an
Adenine to Thymine
Protein mutation of a hydrophilic amino acid to a
hydrophobic one
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Globin Switching
Schechter, 2008
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• Sickle-Cell Anemia is
caused by a mutation in the
adult β chains of the
hemoglobin structure
• Thalassemia is a reduced
production of α/β globin
King, 2009
Can embryonic globins be turned on to take place of dysfunctional
adult globins?
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Krüppel-Like Factor 2, KLF2, is a transcription factor important in
gene expression during development and differentiation.
Vasculature
Lungs
Erythroid morphology and function
* Transcription factors are proteins that initiate or regulate the process of transcription, in which RNA is created from DNA
(Campbell and Reece, 2004)
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Embryonic Day 12.5 (E 12.5) KLF2 null embryos have a yolk sac that lacks blood
They also show growth retardation as compared to wildtype
Wani et al., 1998
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Basu et al., 2005
KLF2 plays part in the
expression of the human
embryonic gene ε in
transgenic mice
KLF2 might have the therapeutic value in treating heminoglobinopathies
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The β-globin locus contains CACCC binding sites in the promoters of the β-like genes, which could serve as targets for KLF2 binding
Erythroid Krüppel-Like Factor, EKLF, binds the β-globin
genes through their CACCC motif
EKLF and KLF2 have high similarities in their zinc fingers
which means that KLF2 might be able to bind to this CACCC
element
Knight and Shimeld, 2001
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Rosenthal and Brown, 2007
This enzyme eliminates targeted sequences by binding to both of the loxP sites and bringing them together to remove the unwanted exon
• Cre can be used under the control of tissue-specific promoters,
deleting genes only in those cells
• Mice with this construct were used in the study to conditionally
knockout the KLF2 in erythroid cells
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• Mating
• Genotyping
• KLF2 mRNA Quantification
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KLF2 F/+, βCre KLF2 F/F
KLF2 F/F, βCre KLF2 F/FKLF2 F/+, βCre
KLF2 F/+
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Experiments
Polymerase Chain Reactions (PCR)
To amplify wanted DNA sequences
Quantitative Reverse Transcriptase-PCR
To check if there is KLF2 mRNA reduction
Phenol-Chloroform Extraction
RNA Isolation
Pitocchelli, 2001
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• Genotyping Results
• KLF2 mRNA Reduction?
• What now?
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Sample Cre Picture
Sample Flox Picture
If βCre is present then a single band will appear, band size: 256 base pairs (bp)
Upper band- +/+, 376 bp Lower band- F/F, ~303 bpBoth bands- F/+
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+ -
+/+
F/F
F/+
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Courtesy of Mohua Basu
Sample #595 can be used for the experiment since it shows significant reduction of KLF2 mRNA
At least 2 more samples are needed
Percent KLF2 mRNA in E 10.5 blood
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If embryonic globin mRNA is:
• Reduced, this would suggest that KLF2 may directly regulate
embryonic globin gene expression by binding to the CACCC motif
• Not reduced, KLF2 might play an indirect role in the expression
of embryonic globin genes in erythroid cells
Data Interpretations
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National Institutes of Health- NIDDK
Virginia Commonwealth University
Dr. Joyce Lloyd
Mohua Basu and other Lloyd lab members
Dr. Suzanne Barbour
Dr. Carolyn Conway
Maura Murphy
Jerry Lingrel, University of Cincinnati (KLF2 F/F mice)
Kenneth Peterson, University of Kansas (βCre mice)
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Questions?