Bryan Castro Joyce Lloyd (Mentor )

Virginia Commonwealth University Dept of Human and Molecular Genetics

• Si ck le 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

• 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?

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)

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 plays part in the

expression of the human

embryonic gene ε in

transgenic mice

KLF2 might have the therapeutic value in treating heminoglobinopathies

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

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

KLF2 F/+, βCre KLF2 F/F

KLF2 F/F, βCre KLF2 F/FKLF2 F/+, βCre

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?

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/+

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?