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 Noguchi Genetic mutation of an Adenine to Thymine Protein mutation of a hydrophilic amino acid to a hydrophobic one

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

Basu et al., 2005 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

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

Rosenthal and Brown, 2007 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

Mating Genotyping KLF2 mRNA Quantification

Mating KLF2 F/+, βCreKLF2 F/F KLF2 F/F, βCre KLF2 F/FKLF2 F/+, β CreKLF2 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 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 bp Both bands- F/ /+F/FF/+

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

 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

 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?