Assessment of gene expression profile of hematopoetic stem cells of the yolk sac isolated from bovine embryos

Abstract

The yolk sac is an embryonic membrane that is crucial for the survival of the embryo during the early stages of pregnancy. It has a complex function in hematopoiesis since it is responsible for the development of the first blood cells and other primitive parts of the circulatory system. The transfer of maternal materials to the fetus is also a probable source of stem cells in which erythrocytes, the first blood cells, express transcription factors that specified the differentiation of these cells into hematopoietic cells. The materials transferred to the fetus include amino acids, vitamins and proteins. To characterize a stem cell, the presence of certain cell surface markers is required. However, there are other markers that cannot be present in order for the cell to be properly characterized as a stem cell. The principal genes expressed in hematopoietic stem cells are: SCL, TAL-1, LMO2, GATA-3, RUNX-1, and CD45. This research aims to analyze the gene expression of hematopoietic cells from embryos at different stages of pregnancy in order to characterize the molecular cultures from the yolk sac samples of bovine embryos. The bovine embryo samples are collected from gravidic uteri at the Barra Mansa abattoir in Ribeirão Preto. The yolk sac will be removed and separated from the fetal membranes. They will then be dissected and cultured in medium specific for hematopoietic stem cell (StemPro®-34 SFM). The cells will be maintained in culture for five days, centrifuged, and stored in TRIzol at -80°C. The total RNA will be extracted using a TRIzol kit (Invitrogen). The RNA will be treated with DNase I (Invitrogen), and then converted into cDNA by RT-PCR. Amplification of the cDNA is performed using pairs of primers for the following transcripts: GATA-3, LMO, RUNX-1, ANXA-5 e GAPDH. PCR will always be used to amplify the specific genes from the cDNA. Before beginning the analysis, the amplification conditions will be tested to determine the best reaction pattern and conditions. The values of gene expression will be expressed in relation to expression of the endogenous gene, GAPDH. (AU)