西南农业学报
西南農業學報
서남농업학보
SOUTHWEST CHINA JOURNAL OF AGRICULTURAL SCIENCES
2010年
5期
1711-1719
,共9页
宋天增%冯静%曾宪垠%杨剑波%杨华%韩猛立%杨井泉%黄新%张红琳%陈永耀%贾斌%石国庆
宋天增%馮靜%曾憲垠%楊劍波%楊華%韓猛立%楊井泉%黃新%張紅琳%陳永耀%賈斌%石國慶
송천증%풍정%증헌은%양검파%양화%한맹립%양정천%황신%장홍림%진영요%가빈%석국경
打点注射%卵巢%转基因兔
打點註射%卵巢%轉基因兔
타점주사%란소%전기인토
Multi-point injection%Ovary%Transgenic rabbit
本研究以绿色荧光蛋白为报告基因,转染兔卵母细胞,探索建立操作方便、效率高、成本低、可定量生产转基因兔的可行性方法.选择14只成年新西兰雌兔,每侧卵巢内分别打点注射0.25 mL 0.5～0.8 mg/mL的质粒,注射后第3日、第16日和第31日进行人工辅助配种;卵巢注射后第3日、第16日和31日随机各取1只雌兔卵巢做冰冻切片,置荧光显微镜下观察绿色荧光;应用PCR 和 Southern 杂交检测转基因新生仔兔阳性率.结果经荧光显微镜观察,卵巢注射后第3日配种的雌兔卵巢冰冻切片及48 h以后的胚胎呈现绿色荧光;卵巢注射报告基因后第3日配种的雌兔后代阳性率最高,PCR 和 Southern 杂交检测结果分别为72.7 %和45.5 %;第31日配种的后代阳性率最低,分别为36.8 %和15.8 %;第16日配种、第3日配种和第31日配种雌兔的阳性后代数量呈递减趋势.卵巢注射后第3日配种,虽然后代阳性率较高,但是其产仔数最少.实验结果表明,用注射器直接对卵巢打点注射外源基因生产转基因兔的方法操作简便、高效,为日后大规模制备一些大型家畜的转基因后代奠定了基础.
本研究以綠色熒光蛋白為報告基因,轉染兔卵母細胞,探索建立操作方便、效率高、成本低、可定量生產轉基因兔的可行性方法.選擇14隻成年新西蘭雌兔,每側卵巢內分彆打點註射0.25 mL 0.5～0.8 mg/mL的質粒,註射後第3日、第16日和第31日進行人工輔助配種;卵巢註射後第3日、第16日和31日隨機各取1隻雌兔卵巢做冰凍切片,置熒光顯微鏡下觀察綠色熒光;應用PCR 和 Southern 雜交檢測轉基因新生仔兔暘性率.結果經熒光顯微鏡觀察,卵巢註射後第3日配種的雌兔卵巢冰凍切片及48 h以後的胚胎呈現綠色熒光;卵巢註射報告基因後第3日配種的雌兔後代暘性率最高,PCR 和 Southern 雜交檢測結果分彆為72.7 %和45.5 %;第31日配種的後代暘性率最低,分彆為36.8 %和15.8 %;第16日配種、第3日配種和第31日配種雌兔的暘性後代數量呈遞減趨勢.卵巢註射後第3日配種,雖然後代暘性率較高,但是其產仔數最少.實驗結果錶明,用註射器直接對卵巢打點註射外源基因生產轉基因兔的方法操作簡便、高效,為日後大規模製備一些大型傢畜的轉基因後代奠定瞭基礎.
본연구이록색형광단백위보고기인,전염토란모세포,탐색건립조작방편、효솔고、성본저、가정량생산전기인토적가행성방법.선택14지성년신서란자토,매측란소내분별타점주사0.25 mL 0.5～0.8 mg/mL적질립,주사후제3일、제16일화제31일진행인공보조배충;란소주사후제3일、제16일화31일수궤각취1지자토란소주빙동절편,치형광현미경하관찰록색형광;응용PCR 화 Southern 잡교검측전기인신생자토양성솔.결과경형광현미경관찰,란소주사후제3일배충적자토란소빙동절편급48 h이후적배태정현록색형광;란소주사보고기인후제3일배충적자토후대양성솔최고,PCR 화 Southern 잡교검측결과분별위72.7 %화45.5 %;제31일배충적후대양성솔최저,분별위36.8 %화15.8 %;제16일배충、제3일배충화제31일배충자토적양성후대수량정체감추세.란소주사후제3일배충,수연후대양성솔교고,단시기산자수최소.실험결과표명,용주사기직접대란소타점주사외원기인생산전기인토적방법조작간편、고효,위일후대규모제비일사대형가축적전기인후대전정료기출.
The purpose of this study was to establish an easily operated,high-efficiency,low-cost method of generating transgenic rabbits.Enhanced green fluorescence protein (EGFP) was used as the reporter gene in order to test the efficiency of our protocol.We choosed fourteen healthy multiparous female New Zealand rabbits (8 to 12 months of age),weighing 3.0 to 3.2 kg,were selected and divided into three groups.Each rabbit received multi-point injections to both sides of the ovaries with EGFP plasmid at concentrations of 0.25 mg (0.5-0.8 mg/mL).Three days after injections,Group 1 females were mated with males,and 16 days after injections,Group 2 females were mated with males,and 31 days after injections,Group 3 females were mated with males.Two days after setting up the mating,one female rabbit from each group was randomly selected and green fluorescence was observed in their ovary tissues.After this,newborn transgenic rabbits were genotyped by PCR and Southern blotting.In Group 1,green fluorescence was observed in the frozen ovary section and embryo 48 hours after mating.Further,the offspring from this group had the highest positive rate for EGFP transgene expression by PCR (78.26 %) and Southern blotting (69.57 %).The offspring from Group 3 had the lowest positive rate by PCR and Southern blotting (41.18 % and 11.76 %,respectively).The number of positive offspring shows a decreasing trend for female rabbits mated on days 16,3,and 31,respectively.Although the offspring from female rabbits had the highest positive rate in Group 1,there were fewer newborn;there were also fewer positive individuals than the offspring from female rabbits mated on day 16.Therefore,we found post-operative day 16 to be the best for mating.The results showed that direct injection of exogenous gene into ovaries is a simple,efficient method to produce transgenic rabbits and provide production transgenic animals with a reference result.
