Karen Moskalik


Construction of cDNA Libraries from Differentiating and Apoptotic Murine Red Blood Cells




The objective of this research was to construct a complementary DNA, or cDNA, library which will allow for the identification of genes involved in normal differentiation of red blood cells. Terminal erythroid differentiation is the process by which a nucleated immature red blood cell (erythroblast) becomes a mature non-nucleated red blood cell (erythrocyte). The principle humoral control of this for the transition is erythropoietin (EPO). In the absence of EPO the erythroblasts will undergo apoptosis (programmed cell death). An in vitro mouse model was used. The mice were injected with an anemia-inducing strain of the Friend virus, and kept for two weeks before they were sacrificed. The spleen cells (FVA cells) were purified with a gravity gradient and cultured either in the presence or absence of EPO. Total RNA and messenger RNA (mRNA) was extracted from both cultures. The mRNA was then used to construct the cDNA libraries according to define orientation strategies. The cDNA molecules were ligated into a vector and packaged into phage particles. The phage particles used to infect Eschericia coli and prepare a library. The libraries were screened by differential hybridization. The differential screening showed the presence of five plaques that were expressed in the presence of EPO and were absent without EPO. The five plaques will be subjected to further studies to determine if any novel genes are present.