Construction of an Xpo7 Protein Expression Plasmid and PCR Focused Array Studies of Cell Cycle Related Genes During Erythroid Terminal Differentiation.

摘要

Erythroid terminal differentiation is a systematic procedure in which erythroblasts expunge their nuclei and form mature erythrocytes. Erythrocytes are known to undergo many physical and biochemical changes during the process of erythroid terminal differentiation and the steps that lead to it. This process must be accompanied by a number of changes in gene expression and studying these changes would help better understand how ETD occurs. Previous studies have shown that Xpo7, an exportin, has shown to be upregulated in FVA cell cultures during the process of ETD. Hence it is believed that this gene must play an important role in the process. Xpo7 is known to shuttle substrate from the nucleus into the cytoplasm. Xpo7 antibodies in the lab currently are a crude serum extract from rabbits that were injected with an Xpo7 specific peptide sequence. Construction of a plasmid that expresses an Xpo7 peptide which can be recognized by the anti Xpo7 antibodies in the lab would prove to be very useful in our studies as it would serve as a means of affinity purifying the antibodies and also function as a positive control. This construction proved to be unsuccessful in two tries wherein on one try the sequencing results returned negative and in the second the cloning of the amplicon into the expression vector failed. Another significant part of this thesis was to identify what cell cycle specific genes could play an important role in the process of ETD. This was carried out using a cell cycle specific PCR array from SAbiosciences. It was found that 18 out of the 84 genes on the array were found to be up regulated and 2 genes were found to be down regulated. This project will serve as a basis for better understanding what genes could be playing a role during the process of ETD and also help future students successfully construct an Xpo7 expression construct.