Measurement of fetal γ-globin expression due to KLF1 being a negative or positive regulator By: Ashria Arora.

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Measurement of fetal γ-globin expression due to KLF1 being a negative or positive regulator By: Ashria Arora

ABOUT γ-GLOBIN GENES AND KLF1 Fetal blood cells Globin- type of red blood cells CACCC promoter region KLF1: Transcription factor Regulates beta globin genes Family of 17 members Binds to CACCC

PURPOSE KLF1 regulation γ-globin genes Positive or negative

THE CELL LINE Past experiment- K562 cells Starting off with a cell that does not contain γ-globin or KLFs, Drosophila cell line, S2. Why use luciferase? Fig 1. Ping et all (2005)

METHODS Plasmid constructs- 1. γLuc 2. KLF1 3. Empty vector

METHODS Transfection and Luciferase assay 1. Electroporation 2. Luciferase assay kit -Control- transfect S2 cells with γLuc and an empty vector -Luminometer- measures the amount of light emitted by the S2 cells

METHODS Western Blotting- insure KLF1 is being expressed in the S2 cells Anti-body EKLF used

DISCUSSION Compare the control cells with KLF1 transfected cells 1. Light emitted with γLuc and KLF1 cDNA is more than the control- KLF1 is a positive regulator OR 2. Light emitted with γLuc and KLF1 cDNA is less than the control- KLF1 is a negative regulator 3. Light emitted is equal to the control, therefore the KLF1 expression construct has no impact on the transcription of γ- globin genes.

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