Production Of Transgenic Mice And Pigs Tissue-specifically Expressing1,3-1,4-β-glucanase

1,3-1,4-β-glucan, referred to as β-glucan, is an anti-nutritional factor in cerealplant material, and has bad effects on animal digestive absorption. Although the1,3-1,4-β-glucanase, referred to as β-glucanase, is able to the decompose β-glucan,β-glucanase dose not exist in monogastric animal gastrointestinal tract, and so β-glucan would not be digested in them. Therefore, the new research strategies whichsolve the problem are to produce heterologous enzymes in the animal digestive tractto improve feed utilization rate by generating transgenic animal. In this study,β-glucanase gene fragment was amplified using PCR, and the parotid gland andintestine tissue-specific expressing β-glucanase vector were constructed. Thetransgenic mice expressing β-glucanase in parotid gland and intestine tissue weresuccessful prepared by microinjection method. The transgenic mice were detected atthe DNA, mRNA levels and enzyme activity in order to comprehensively assess thefeasibility of reducing feed β-glucan anti nutritional effect by producing β-glucanasetransgenic pigs. The linearized β-glucanase gene parotid gland and intestinetissue-specific expressing vector were transfected into the porcine fibroblasts andobtained the transgenic cell lines stably expressing β-glucanase. The two kinds of celllines were used as nuclear donors for nuclear transfer technology, and transgenic pigswere produced using cloning method. Finally,3transgenic cloned pigs expressingspecifically β-glucanase in parotid gland tissue and3transgenic cloned pigsexpressing specifically β-glucanase in the intestinal tissue were obtained, respectively.The results were as follows:1. The parotid gland tissue-specific expressing β-glucanase vector was successfullyconstructed. The transgenic mice were produced by the pronuclear microinjection.Both PCR and Southern blotting analysis showed that the mice carried theβ-glucanase gene and the β-glucanase gene would be stably inherited. RT-PCR andNorthern blotting analysis indicated that β-glucanase gene was specifically expressedin the parotid gland tissue. The β-glucanase activity in the saliva was found to be0.18±0.02U/mL. Mice were fed a diet containing2%β-glucan for nutrition metabolism test. The results showed that the average daily gain of transgenic and non-trangenicmice was0.0024g/d and-0.0346g/d, repectively. The crude protein, crude fatdegestibility of transgenic mice were increased by12.15%and5.11%(p<0.05),respectively, compared with that of the non-transgenic mice. While the moisture infeces showed no significant difference between transgenic and non-transgenicmice(p>0.05). These results suggested that foreign β-glucanase were successfullyexpressed in the animal parotid of mice and could reduce the anti-nutritional effect ofβ-glucans in feed.2. Landrace porcine fibroblasts were successfully transfected bypPSPBGPneo-GLU vector harboring β-glucanase gene. By G418screening, thistransgenic cell lines were finally obtained and could specifically expressedβ-glucanase gene in parotid gland tissue and this kind of cell line were used as nucleardonors for nuclear transfer technology. Finally,3landrace transgenic cloned pigsexpressing specifically β-glucanase in parotid gland tissue were obtained. PCR andSouthern blotting analysis showed that the pigs carried the β-glucanase gene. Theβ-glucanase activity in the saliva was found to be3.2U/mL、0.07U/mL and0.03U/mL in the pigs. The metabolism test showed the energy, crude protein, crude fatdegestibility of transgenic pigs were increased by (15.09%,6.20%and9.09%),(20.73%,5.41%and1.76%) and (39.53%,5.99%and4.19%)compared with that ofnon-transgenic pigs, respectively. While the moisture in feces were reduced by16.10%,6.74%and2.08%, respectively. These results suggested that foreignβ-glucanase were successfully expressed in the animal parotid of pig and could reducethe anti-nutritional effect of β-glucans in feed.3. The β-glucanase gene was cloned into a intestine specific expressing vector withMUC2promoter (MUC2-GLU-LV). The transgenic mice were prepared bymicroinjection. PCR and Southern blotting analysis showed that the mice carried theβ-glucanase gene. Northern blotting analysis indicated that β-glucanase wasspecifically expressed in the intestine of the transgenic mice. The β-glucanase activityin the intestinal juice was found to be1.23±0.12U/mL. Mice were fed a dietcontaining2%β-glucan for nutrition metabolism test. The results showed that theaverage daily gain of transgenic and non-transgenic mice was0.058g/d and-0.024g/d, respectively. The crude protein, crude fat degestibility of transgenic mice wereincreased by9.32%and5.09%(p<0.05), respectively, compared with that of the non-transgenic mice. While the moisture in feces were reduced by12.16%(p<0.05).These results suggested that foreign β-glucanase were successfully expressed in theintesteine of mice and could reduce the anti-nutritional effect of β-glucans in feed.4. Landrace porcine fibroblasts were successfully transfected by MUC2-GLU-LVvector harboring β-glucanase gene. By G418screening, this kind of transgenic celllines were finally obtained and could specifically expressed β-glucanase gene inintestine tissue, and this kind of cells were used as nuclear donors for nuclear transfertechnology. Finally,3landrace transgenic cloned pigs expressing specificallyβ-glucanase in the intestine tissue were obtained by PCR analysis. Additionaldetection is under way.It is the first to produce the transgenic mice expressing β-glucanase gene inparotid gland and intestine tissue in the study. The results demonstrated that theanti-nutritional effects of β-glucan could be effectively reduced. Transgenic pigstissue-specifically expressing β-glucanase were obtained, which provided thefoundation for improving the rate of digestion and the establishment ofenvironment-friendly transgenic pigs.