Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

Advances in the generation of transgenic pigs via embryo-derived and primordial germ cell-derived cells.

The development of new technologies that would increase the efficiency for generation of transgenic livestock and would overcome some of the problems associated with random insertion of the transgene will greatly benefit animal agriculture. A potential alternative technology to pronuclear injection for the generation of transgenic pigs involves the isolation, culture and genetic manipulation of cell lines that can be reintroduced into the embryo for participation in the formation of the germ cells. We have isolated and cultured pig primordial germ cells (PGC) while maintaining them in an undifferentiated state as determined by morphology and alkaline phosphatase (AP) activity. More importantly, PGC-derived cells were stably transformed with the green fluorescent protein marker driven by the cytomegalovirus promoter. After visual identification of transgenic colonies, the pluripotential characteristics of the transgenic PGC-derived cells were tested by chimaera formation and to date we have identified, by genomic Southern blots, two chimaeric fetuses that contain tissues with the transgene incorporated into their chromosomes. To our knowledge, this is the first report of a chimaeric transgenic pig fetus obtained via a cultured cell line.

Generation of transgenic porcine chimeras using primordial germ cell-derived colonies.

In mice, two pluripotent cell lines, embryonic stem (ES) cells and embryonic germ (EG) cells, have been identified. We present here results indicating that porcine EG cell lines can be isolated, genetically transformed, and utilized to make transgenic chimeras. Briefly, primordial germ cells (PGCs) were isolated from Day 25-27 fetuses and plated on STO feeder cells in Dulbecco's modified Eagle's medium:Ham's F-10 medium supplemented with 0.01 mM nonessential amino acids, 2 mM glutamine, 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 40 ng/ml human stem cell factor, 20 ng/ml human basic fibroblast growth factor, and 20 ng/ml human leukemia inhibitory factor. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein under control of the cytomegalovirus promoter. After electroporation, cells were plated and later examined under fluorescein isothiocyanate excitation. Fluorescent colonies were selected for chimera generation. Blastocysts collected from gilts on Day 5 were injected with 10-15 transgenic PGC-derived cells and transferred into recipient gilts. Gilts were hysterectomized on Day 25, and fetal tissues were analyzed by Southern blotting. Three chimeras out of 20 fetuses analyzed were transgenic. Additionally, when one recipient gilt was allowed to go to term, one piglet with transgenic contribution was identified.

Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion.

The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadec anoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.