Expression Of Surfactant Protein B In Mice Derived Completely From Embryonic Stem Cells

Mouse embryonic stem (ES) cells and tetraploid embryos have different distribution. The yolk sac endoderm and the trophoblast cell lineages are tetraploid embryo-derived, whereas the embryo proper, the amnion, the yolk sac mesoderm, the allantois, and the chorionic mesodermderived part of the placenta are completely ES cell-derived. Thus, we can obtain the ES mouse completely from ES cells with the method of tetraploid embryo complementation.Many clone animals died of respiratory distress including ES mouse. The respiratory distress was the main disease resulting in death in neonatal ES mouse. The life span of those animals was less than an hour. The clinical and pathological features of respiratory distress are similar to neonatal respiratory distress syndrome (NRDS) in clinical pediatrics. NR.DS results from insufficient levels of pulmonary surfactant protein(SP). Pulmonary surfactant protein is essential for the expansion of the alveoli in the lungs, and its deficiency causes the alveoli to collapse, preventing the infants from breathing properly. So far, four SP have been found in mouse: SP-A, SP-B, SP-C and SP-D, in particular SP-B, have been shown to be critical for normal alveolar function. Transgenic newborn mice lacking the SP-B gene fail to inflate their lungs and die of respiratory distress shortly after birth, so we deduce that respiratory distress maybe mainly resulting from abnormal expression of SP-B in neonatal clone animals.Genomic DNA was extracted from 12 tissues (lung, liver, brain, pancreas, intestine, bladder, stomach, heart, testis or ovary, spleen, kidney, muscle) of adult ES mice and from 4 tissues(heart, liver, brain, muscle) of newborn ES mice died of respiratory distress. PCR amplification of five microsatellite DNA loci, D2Mit493, D10Mit134, D16Mit189, D5Mit138 and D5Mit346 to detect the tetraploid embryo contribution in ES mice to determine whether these ES mice are derived completely from ES cells. The data suggest that ES mice are derived completely from ES cells.Lungs were collected from one of the neonatal ES mice with respiratory distress and a normal neonatal mouse and submitted for routine histopathological analysis. Histological examination of the lungs revealed extensive atelectasis, alveoli collapse, and thickened alveolar wall in the ES mice with respiratory distress, whereas the lungs of normal neonatal mice were fully inflated and the alveoli were expanded. Lung proteins were extracted respectively form 12 adult ES mice, 6 nenotal ES mice died of respiratory distress and control mice.Then we detected the expression of SP-B with western blot. We observed weaker SP-B expression in the adult ES mice than in the normal adult mice . Similarly, we observed no SP-B expression in the neonatal ES mice with respiratory distress and slight SP-B expression in the normal neonatal mice .These data suggest that severe lacking SP-B may be the key reason resulting in respiratory distress, however the survival won't been affected by moderate decrease of SP-B in ES mice.