Transcript Genotyping of Mice to Study Role of Krüppel

Bryan Castro
Joyce Lloyd (Mentor )
Virginia Commonwealth University
Dept of Human and Molecular Genetics
• Sickle Cell Anemia
• Hemoglobin
• KLF2
• β-Globin Promoter
• Cre Recombinase
Sickle Cell Anemia
 Hemoglobin that sickles
 Inherited Hemoglobinopathy
 Predominant in people of African or Mediterranean
ancestry
Sickle Cell Anemia
Genetic
mutation of an
Adenine to
Thymine
Protein mutation
of a hydrophilic
amino acid to a
hydrophobic one
Noguchi
Globin Switching
Schechter, 2008
Hemoglobin
• 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?
KLF2
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)
KLF2 -/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
KLF2 and Human ε
KLF2 plays part in the
expression of the human
embryonic gene ε in
transgenic mice
KLF2 might have the therapeutic value in treating
heminoglobinopathies
Basu et al., 2005
β-Globin Promoters
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
Cre Recombinase
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
Rosenthal and Brown, 2007
• Mating
• Genotyping
• KLF2 mRNA Quantification
Mating
KLF2 F/+, βCre
KLF2 F/+, βCre
KLF2 F/F, βCre
KLF2 F/F
KLF2 F/F
KLF2 F/+
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
• Genotyping Results
• KLF2 mRNA Reduction?
• What now?
Genotyping
Sample Cre Picture
If βCre is present then a
+ -
single band will appear,
band size: 256 base pairs
(bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
+/+
Upper band- +/+, 376 bp
Lower band- F/F, ~303 bp
Both bands- F/+
F/F
F/+
Sample Flox Picture
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Percent KLF2 mRNA in E 10.5
blood
140
Percent of KLF2 mRNA/ GPA
120
Sample #595 can be
used for the
experiment since it
shows significant
reduction of KLF2
mRNA
100
80
60
40
20
0
+/+
630
631
634
594
600
F/F
601
606
607
608
595
F/F Cre
Embryo Samples
Courtesy of Mohua Basu
At least 2 more
samples are needed
Data Interpretations
 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
 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)
Questions?