The Study of CMAH Gene-Edited Mutant Piglets without Expressing Neu5GC Resistant to Porcine Epidemic Diarrhea Virus Infection
| Autor: | Kai-Xuan Hsiao, 蕭凱烜 |
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| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 107 Porcine epidemic diarrhea virus (PEDV) was first recognized in Europe since 1971, outbreaks of the disease also occurred in Taiwan at the end of 2013. Pigs infected by PEDV have been detected clinical sign including diarrhea and vomiting, and the morality rate of PEDV is up to 100% in neonatal piglets. The whole process of the infection of virus, first virus which bind with putative receptors such as sialic acid and aminopeptidase N(APN) to contact cells, leading infected cells gradually and continuously into necrosis, shedding and villi atrophy. Then atrophy causes the infected piglets uncapable absorbing nutrients, water and minerals, resulting in losing body weight and death. PEDV has resulted in significant economic losses in pig production in Taiwan and also in Japan, Korea, China and America, and furthermore still no effective vaccines against PEDV infection. The purpose of this study was to exam PEDV resistance of CMAH mutant (knockout, KO) piglets which generated by CRISPR/Cas9 gene editing techniques. Cytoplasm of pronuclear porcine new fertilized eggs were microinjected with two sites of sgRNA/Cas9 mRNA, and four alive piglets (L667-02,-10,-11 and -12) were born and all bi-alleles mutants. Their tissues were screened and shown without Neu5GC, and also germline transmission to their offspring. All piglets used in this study were neonatal F1 CMAH KO offspring, which delivered by the three female founders (L667-10,-11,-12) served by the male founder (L667-02). The 2-day-old or 3-day-old KO piglets were used for Exp. I and Exp. III or Exp. II, respectively; and the concurrently same age wild type (WT) piglets were used as control. Piglets were oral inoculated with 1 mL of 1x103 TCID50/mL PEDV. Then at every 4 h for three days, hand-feed milk and clean water were offered and their clinical signs were recorded for vitality analysis. After 72 h PEDV challenged, all piglets were sacrificed for necropsy, and the upper and middle of jejunum and ileum were sampled for immunofluorescence assay (IFA). Results showed that all of the piglets appeared typical clinical signs including diarrhea, vomiting and losing weight. In Exp. I, four of six KO piglets and all of six WT piglet died (67% vs. 100%), survival rate didn’t have a significant difference between two genotypes of piglets though the high variation in the extent of pathologic changes. The KO piglets apparently have a better resistance to PEDV than WT piglets. Nevertheless, in Exp. II, resulted no significant difference in clinical data, similar to Exp. I, and even in pathologic information. In Exp. III, in order to rulling out the interferece of Neu5GC/Neu5AC in sow milk and commercial baby milk powder, which used in Exp. I and II, piglets were fed dam’s milk or skim milk for one day, and then replaced to lactated Ringer’s solution supplementary with 5% glucose. Results indicated that the survival rate between both genotypes of piglets were all improved and no significant difference. Moreover, after analyzing by HPLC, commercial baby milk powder and WT dam’s milk didn’t contain a high quantity of Neu5GC, which not appeared in KO sow’s milk. The results suggest that milk isn’t the key factor in interfere the result. Furthermore, orally offer made almost the piglets survive that could be an example for supportive play chotherapy on infected piglets. This study demonstrates that CMAH gene editing, piglets without Neu5GC expression have not increased resistance to PEDV infection, but may lessen the severity of the infection and delay its occurence. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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