To elucidate the in vivo function of GATA-1 during hematopoiesis, we specifically disrupted the erythroid promoter of the GATA-1 gene in embryonic stem cells and generated germ line chimeras. Male offspring of chimeras bearing the targeted mutation were found to die by 12.5 days post coitus due to severe anemia while heterozygous females displayed characteristics ranging from severe anemia to normal erythropoiesis. When female heterozygotes were crossed with transgenic males carrying a reporter gene, which specifically marks primitive erythroid progenitors, massive accumulation of undifferentiated erythroid cells were observed in the yolk sacs of the GATA-1-mutant embryos, demonstrating that GATA-1 is required for the terminal differentiation of primitive erythroid cells in vivo.

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Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling-time of about 22-31 hr, but did not grow at 39 degrees C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma-glutamyltranspeptidase activity in TR-BBBs was approximately 13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 +/- 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.

The AML1 and PEBP2b/CBFb genes encode the DNAbinding and non-binding subunits, respectively, of the heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype, namely the complete lack of de®nitive hematopoiesis in the fetal liver on embryonic day 11.5 (E11.5). We examined and compared the expression levels of various hematopoiesis-related genes in wild type embryos and in embryos mutated for AML1 or PEBP2b/CBFb. The RNAs were prepared from the yolk sacs of E9.5 embryos, from the aorta-gonad-mesonephros regions of E11.5 embryos and from the livers of E11.5 embryos and RT ± PCR was performed to detect various gene transcripts. Transcripts were detected for most of the hematopoiesis-related genes that encode transcription factors, cytokines and cytokine receptors, even in tissues from homozygously targeted embryos. On the other hand, PU.1 transcripts were never detected in any tissue of AML1(7/7) or PEBP2b/CBFb(7/7) embryos. In addition, transcripts for the Vav,¯k-2/¯t-3, M-CSF receptor, G-CSF receptor and c-Myb genes were not detected in certain tissues of the (7/7) embryos. The results suggest that the expression of a particular set of hematopoiesis-related genes is closely correlated with the PEBP2/CBF function.

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