頁籤選單縮合
題名 | Generation of GGTA1 Knockout Pigs by Using Direct Pronuclear Microinjection with TALEN Plasmid DNA Vectors=直接原核顯微注射TALEN基因剔除質體DNA產製GGTA1基因剔除豬 |
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作者姓名(中文) | 莊景凱; 陳建宏; 蘇郁秀; 彭書慧; 林岱昀; 黃仲琳; 楊天樹; 杜清富; | 書刊名 | 中國畜牧學會會誌 |
卷期 | 45:3 2016.09[民105.09] |
頁次 | 頁223-240 |
分類號 | 368.4 |
關鍵詞 | 性腺傳承; GGTA1基因剔除; 原核顯微注射; TALEN質體DNA; Germline transmission; GGTA1 knockout pigs; Pronuclear microinjection; TALEN plasmid DNAs; |
語文 | 英文(English) |
英文摘要 | The shortage of human allografts has promoted attempts to produce genetically engineered (GE) pigs with an aim to harvest xeongrafts with minimum rejections. Recently, zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/associated endonuclease (CRISPR/Cas) combined with somatic cell nuclear transfer (SCNT) procedures have been used to generate knockout (KO) pigs to overcome xeno-rejections caused by -Gal and non-Gal. We have cloned porcine GGTA1 KO vectors by using TALEN toolbox; but whether GGTA1 KO pigs could be generated through direct pronuclear microinjection with cloned plasmid vectors has not yet been known, and it was therefore tested in the present study. A total of 65 fertilized eggs were directly microinjected with the KO plasmid vectors and subsequently transferred into two foster dams. According to EcoRV PCR/RFLP and PCR product sequencing, two male GGTA1 KO piglets were identified among all 13 farrowed with one bi-allelic KO (bKO) and the other a mono-allelic KO (mKO). The bKO was a mosaic carried one allele with 11 bp deletion and the other allele with 2 bp (AT) insertion. The peripheral blood mononuclear cells (PBMCs) studies, conducted by IB4-lectin staining and flow cytometry analyzing, revealed a significant reduction in surface-Gal expression in bKO but not in mKO pig. Both KO pigs were brought up to adolescence, and their sperm DNAs were ana lyzed and results of EcoRV PCR/RFLP indicated that germ cells of bKO pig were either genomic deletion or insertion; while those of mKO were a mixture of deletion and wild type. Insemination of bKO semen to normal sows successfully produced four litters of 29 piglets of which 11 were identified by PCR products/TA-cloned sequencing as genomic deletion and the other 18 were AT insertion. These data suggested that TALEN KO plasmid vector could be directly microinjected into porcine fertilized eggs to generate mono-or bi-allelic KO pigs with normal germline transmission capability. Generation of GE pigs for xenotransplantation or any other purpose will be facilitated by adopting the one step approach reported herein. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。