Generation Of Lung Deficient Pigs By Overexpressing Dominant Negative FGFR2

Human organ transplantation has improved duration and quality of life for many people, but its full potential is critically limited by short supply of available organs. One solution is xenotransplantation, although this comes with its own set of challenges. Using pig lung donors, many labs have been working on lung transplants into baboons as a surrogate for a human recipient. Lungs in particular are multiple immune rejection mechanisms, during the transplantation process generally. In this experiment, generation of lung deficient pigs with somatic cell nuclear transfer (SCNT) technology, and inject PSCs of patients into porcine blastocysts to produce a humanized lung.Lung development begins when two lung buds sprout from the epithelium of the embryonic gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of the two lung buds, a process called branching morphogenesis. FGFR2b is expressed in the epithelium of a number of embryonic organs including the lung buds. To block the function of FGFR2b during branching morphogenesis of the lung without affecting its function in other embryonic tissues, the human surfactant protein C promoter was used to target expression of a dominant negative FGFR2c and FGFR2b and soluble dominant-negative mutant FGFR2b-HFc exclusively to lung bud epithelium in transgenic pig. We expect the dominant negative FGF receptor block airway branching and epithelial differentiation.ObjectiveTo generate lung deficient pigs by blocking the function of FGFR2b.Methods1. Three expressed dominant negative DNFGFR2 plasmid were designed and constructed. one expressed DNFGFR2c lack of tyrosine kinase domain, and anther expressed DNFGFR2b lack of tyrosine kinase domain, last one expressed a soluble dominant-negative mutant FGFR2b-HFc with FGFR2b extracellular region and the pig IgG heavy chain hinge region and constant region.2. The plasmids were transfected into porcine primary fetal fibroblasts, cell colonies formed under Puromycin selection. Cell colonies were screened and collected for PCR.3. With the aid of somatic cell nuclear transfer (SCNT) technology, cell pools were used as nuclear donors to produce cloned DNFGFR2c、DNFGFR2b and DNFGFR2b-HFc transgene piglets.4. For cloned piglets, PCR sequencing was used to identify their genotypes; RT-PCR was taken for the detection of mRNA expression; the organizational structure of lung was detected by HE staining; Immunohistochemistry was also performed on lung development.ResultsBy applying the SCNT technology, three kinds of cloned pigs were constructed. The genotypes of the cloned piglets were all detected as FGFR2 transgene. To detect the DNFGFR2 protein expression of these gene-mutation piglets, RT-PCR was performed using mRNA extraction of lung tissue. All of the cloned piglets expressed DNFGFR2 mRNA.Four DNFGFR2c recipient gilts were pregnant.one was terminated at E43 to collect fetuses for PFF isolation and phenotype analysis. Three normal fetuses were collected. The other three recipient was maintained until term and delivered seven piglets, four died at perinatal period, two died at neonatal period one died a month after birth. Compared to the WT piglet, neonatal DNFGFR2c transgene piglets were high mortality (6/7), lung tissues were developmental normal.Two DNFGFR2b recipient gilts were pregnant. one was terminated at E43. Three normal fetuses were collected. The other one recipient was maintained until term and delivered three piglets, E43 DNFGFR2b piglets showed that lung consisted predominantly of mesenchymal tissue, fewer differentiated bronchial epithelium. Peripheral regions of DNFGFR2b lung consisted predominantly of mesenchymal tissue with small amount bronchial tube. E43 DNFGFR2b piglets showed that lung consisted predominantly of big alveoli.Two DNFGFR2b-HFc recipient gilts were pregnant, one was terminated at E43. Six normal fetuses were collected. The other one recipient was maintained until term and delivered six piglets, E43 DNFGFR2b-HFc piglets showed that lung consisted predominantly of mesenchymal tissue, fewer differentiated bronchial epithelium. Peripheral regions of DNFGFR2b-HFc lung consisted of mesenchymal tissue with no bronchial tube. E110 DNFGFR2b-HFc piglets showed that lung consisted predominantly of small immature alveoli.ConclusionOur study indicates that the overexpression of DNFGFR2c、DNFGFR2b and DNFGFR2b-HFc on the surface of epithelial cell blocked the function of FGFR2b and generated lung-deficient pigs. Overexpressed DNFGFR2b and DNFGFR2b-HFc pigs can developed lungs with more severe dysgenesis of lung than DNFGFR2c.