CN1528131A - 不育转基因鱼的制备方法 - Google Patents
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Abstract
本发明公开了一种不育转基因鱼的制备方法，以鲤鱼肌动蛋白基因启动子作为启动子，以反义的286bp鲤鱼鲑鱼型促性腺激素释放激素基因互补脱氧核糖核酸片段[包括sGnRH十肽、促性腺激素释放激素相关肽编码区和64bp的3′非编码区序列]为靶位基因，草鱼生长激素基因3′侧翼序列为终止顺序，构建鲑鱼型促性腺激素释放激素反义RNA表达载体，采用显微注射法将重组DNA片段导入鲤鱼受精卵，聚合链式酶反应及放射免疫检测筛选获得不育转基因鱼。本发明方法易行，操作方便，为解决转基因鱼的遗传和生态安全问题提供了具有普遍适用性的技术平台。
Description
技术领域
本发明涉及转基因鱼研究领域，更具体涉及一种不育转基因鱼的制备方法。
背景技术
1984年，中国科学院水生生物研究所在世界上率先开展了鱼类基因工程定向育种研究，建立了完整的转基因鱼理论模型和完善的实验技术体系(Zhu，et al，1985；朱作言等，1989)。在此基础上，快速生长的转“全鱼”生长激素基因鲤鱼研究取得重要进展，规模化养殖试验显示，转基因鲤鱼子代群体平均体重比对照鱼增重快42％，饵料利用效率提高18.5％，池塘养殖综合经济效益提高125.7％(汪亚平等，2001；朱作言等，2001)。与此同时，欧、美等国在快速生长转基因虹鳟、大西洋鲑和罗非鱼等研究方面亦取得重要进展，规模化养殖试验也在进行中，但迄今尚无转基因鱼商品化实例。
诚然，作为一种定向遗传改良的生物体，转基因鱼的生物安全性受到了全社会的普遍关注。转基因鱼的生物安全性包括作为食物的消费安全、释放的遗传安全和生态安全。目前，转基因鱼研究均采用鱼类来源甚至自身来源的基因元件，极大地消除了转基因鱼的食品安全隐患；而且转“全鱼”基因鲤鱼的食品安全性检测结果也表明，摄食转基因鱼对小鼠的生长、发育和生殖等均无显著影响(张甫英等，2000)。高效养殖模式的建立和食品安全性研究结果的获得，为转基因鱼的商品化奠定了基础。因此，遗传和生态安全及其控制技术已成为转基因鱼研究亟待解决的问题，也是转基因鱼实现产业化的必要前提。转基因鱼的遗传和生态安全问题概括起来包括两个方面：其一，转基因鱼可能与野生种和近缘物种杂交，造成自然种质资源基因库污染；其二，转基因鱼可能形成优势种群，破坏水生态系统的群落结构，造成难以逆转的影响。而通过制备不育的转基因鱼，一方面可以控制转植基因通过有性交配的方式发生遗传漂移，另一方面，可以防止逃逸到自然水体中的转基因鱼形成优势种群，进而对水生态系统产生不利影响。基于此，转基因鱼的遗传和生态安全可以通过对转基因鱼的生殖控制来解决。
目前不育转基因鱼的培育技术是采用三倍体策略，通过鱼类倍间杂交的方法进行的，即将二倍体的转基因鱼与四倍体鱼杂交，从而获得不育的三倍体转基因鱼。但是，四倍体鱼的获得极其困难，我国学者自行制备的异源四倍体鲫鲤是目前世界上唯一一例能自身繁殖、遗传性状稳定且没有经过化学或物理方法处理获得的四倍体鱼种群(刘筠，1993)。因此，采用三倍体策略制备不育转基因鱼的应用受到很大限制，只能是解决转基因鱼遗传生态安全问题的一个特例，不具备普遍性。
发明内容
本发明的目的在于提供一种不育转基因鱼的制备方法，该方法简便，操作方便，通过反义核酸技术阻遏鲤鱼性腺发育及性成熟相关基因----鲑鱼型促性腺激素释放激素基因(sGnRH)的表达，阻抑转基因鱼的生殖功能，从而获得不育的转基因鱼，为解决转基因鱼的遗传和生态安全问题提供具有普遍适用性的技术平台。
为了达到上述目的，本发明采用了以下技术措施：根据反义核酸作用原理，构建反义RNA表达载体，遏制鲤鱼鲑鱼型促性腺激素释放激素(sGnRH)基因的表达，制备获得不育的转基因鱼。其步骤是：A：以鲤鱼肌动蛋白(β-actin)基因的启动子为启动子，以反义的286bp鲤鱼鲑鱼型促性腺激素释放激素基因(sGnRH)的cDNA片段为靶位基因[包括sGnRH十肽、促性腺激素释放激素相关肽(GAP)编码区和64bp 3′非编码区序列]，以草鱼生长激素基因的3′侧翼顺序为终止顺序，构建反义RNA表达载体pCAsGnRHc-antisense。B：经限制性内切酶(Hind III和EcoR I)消化，显微注射法将重组基因CAsGnRHc-antisense导入鲤鱼受精卵中。C：采用PCR筛选转重组基因的阳性鱼，进而通过放射免疫检测获得不育转基因鱼。
本发明与现有技术相比，具有以下优点和效果：提供了一种制备不育转基因鱼的新的技术平台，采用该技术，不用培育四倍体鱼，可以直接获得不育的转基因鱼，不育转基因鱼出现的频率约32％。该方法易行，操作简便，不受培育四倍体鱼的限制，具有普适性，能广泛应用于转基因鱼育种实践，进而为从根本上解决转基因鱼的遗传和生态安全的问题提供了具有普遍适用性的技术平台。
附图说明
图1为鲤鱼鲑鱼型促性腺激素释放激素基因(sGnRH)反义RNA表达载体(pCAsGnRHc-antisense)示意图。
1：鲤鱼肌动蛋白(β-actin)基因的启动子，长2.5kb；
2：反向的鲤鱼鲑鱼型促性腺激素释放激素基因(sGnRH)cDNA片段，长286bp，包括鲑鱼型促性腺激素释放激素基因(sGnRH)十肽、促性腺激素释放激素相关肽(GAP)编码区和64bp 3′非编码区序列.
3：草鱼生长激素基因3′侧翼顺序，长0.8kb；
4：质粒载体pUC118，长3.2kb。
5：限制性内切酶Hind III位点。
6：限制性内切酶EcoR I位点。
具体实施方式
其步骤是：
A，鲑鱼型促性腺激素释放激素反义RNA表达载体(pCAsGnRHc-antisense)的构建
在鲑鱼型促性腺激素释放激素反义RNA表达载体(pCAsGnRHc-antisense)中，鲤鱼肌动蛋白(β-actin)基因的启动子2.5kb片段为启动子，按3′--5′的方向，反向的286bp鲤鱼鲑鱼型促性腺激素释放激素(sGnRH)基因的cDNA片段为靶位基因插入在启动子的下游，随后的草鱼生长激素基因的3′侧翼顺序0.8kb为终止顺序，反义载体骨架为pUC118。
B，转基因鱼的制备
将鲑鱼型促性腺激素释放激素反义RNA表达载体(pCAsGnRHc-antisense)分别用限制性内切酶(EcoR I和Hind III)消化，得到线性重组DNA片段(CAsGnRHc-antisense)。回收的DNA溶于缓冲液ST(88mM NaCl，10mM Tris-HCl，pH7.5)，调节其终浓度为50ng/μL，鲤鱼受精卵用0.25％的胰蛋白酶消化去除卵膜，采用显微注射的方法，在第一次卵裂前将DNA溶液导入受精卵动物极，DNA注射剂量约5×105拷贝/卵，完成显微操作后的受精卵置于实验室无菌环境中孵化。
C，不育转基因鱼的筛选
剪取转CAsGnRHc-antisense基因鱼尾鳍少许，提取DNA，采用PCR技术检测转基因阳性鱼；从转基因阳性鱼尾部血管取血少许，检测转基因阳性鱼血清中11-酮基睾酮(11-ketotestosterone)的浓度，确定雄性鲤鱼，同时采用双抗体放射免疫法(Radioimmunoassay，RIA)测定血清的促性腺激素(Gonadotropin，GtH)含量，若其血清促性腺激素(GtH)含量低于同批转基因阳性鱼促性腺激素(GtH)含量的60％，则筛选获得不育的转基因鱼。其中，正向检测引物(5′-CCATGGCGTATCGATGTCGAC-3′)位于启动子与反向靶向序列之间的接头序列上，反向检测引物(5′-CATGGCTTTGCCAGCATTGG-3′)位于靶向序列上，两引物扩增的片段长度为328bp。
SEQUENCE LISTING
<110>中国科学院水生生物研究所
<120>不育转基因鱼的制备方法
<130>不育转基因鱼的制备方法
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<220>
<221>3’UTR
<222>(2859)..(3645)
<223>
<400>1
aagcttttag accttcttac ttttggggat tatataagta ttttctcaat aaatatctca 60
tatcttactg tggtttaact gctgaatcta aaattttaat acaaaagtag ttatatttgt 120
tgtacattgt aaactataac ttaacttcag tttcagagaa actcatgtgc tcaaaatgta 180
aaaaaagttt cctgttaaat attttgtaaa tgtattgaag acaaaataag aaaaaaaaaa 240
tataagccac taaatcacac tgtccttggt atcagcaaga gattctgaca taatcagctg 300
tttttgttta ttactgccat tgaaggccat gtgcattagt cccaagttac acattaaaaa 360
gtcacatgta gcttaccaac atcagtgctg ttcaagcaca gcctcatcta ctattcaaac 420
tgtggcacca tctaaaatat gccagaattt ttttatttaa tgaatttgac cctgaaatat 480
gtattaatat cactcctgtg atttttttgt aatcagctta caattacagg aatgcaagcc 540
tgattcatta caagtttcac tacactttct ctgacaacat cacctactga actcagacca 600
gctagttgct ccttaagtat acaatcatgt cactaatcct catttcaatg aaaaataccc 660
ctattgtact tggtacttgg tagataacca cagagcagta ttatgccatt attgtgaata 720
caataagagg taaatgacct acagagctgc tgctgctgtt gtgttagatt gtaaacacag 780
cacaggatca aggaggtgtc catcactatg accaatacta gcactttgca caggctcttt 840
gaaaggctga aaagagcctt attggcgtta tcacaacaaa atacgcaaat acggaaaaca 900
acgtattgaa cttcgcaaac aaaaaacagc gattttgatg aaaatcgctt agggatcccc 960
ccttgctctt caaacaatcc agcttctcct tctttcactc tcaagttgca agaagcaagt 1020
gtagcaatgt gcacgcgaca gccgggtgtg tgacgctgga ccaatcagag cgcagagctc 1080
cgaaagttta ccttttatgg ctagagccgg gcatctgccg tcatataaaa gagcgcgccc 1140
agcgtctcag cctcactttg agctcctcca cacgcagcta gtgcggaata tcatctgcct 1200
gtaacccatt ctctaaagtc gacaaacccc cccaaaccta aggtgagttg atctttaagc 1260
tagcttttta cattttcagc tcgcatatat caattcgaac gtttaattag aatgtttaaa 1320
taaagccaga ttaaatgatt aggctcagtt accggtcttt tttttctcat ttacgtgcga 1380
actctgctta aactctagtt attctttatt aatatgtggt tatttttata tatgtatgtg 1440
ttatcataac tgtactggct atgtcaggtg gtaatgactg taacgttacg ttactcgttg 1500
taggcacgac attgaatggg ccggtgttga aataagtctt caaccccttt taacctcaaa 1560
atgtgctctg gttaacaagg attttaacag ctatcagtat gactgtgcgg ttttaaagcc 1620
gttagtgagg cacgttgcac acttgatgga tggccggaat gggaagttct ttatgcaggc 1680
agtgctgcag cagggtgtga cctactttag ctaacgttag ccggctaacc agcattcatc 1740
tgccggtaac ttgagtctaa tattctctca tgtgatatcg aagtgatcaa agacacgtct 1800
gttagctcac tttaaccaac tgtagtgaaa aatagcgcag tgtgcagccc ttcagtcttt 1860
catttaggct gattattcaa tcattttatt aactattaac gcgttactaa acgtaaggta 1920
acgtagtcag tttttaataa ctggtgaaaa gtactggttg ggtttaaatg gtgacttata 1980
attgtgttgg agggggaaac ctttttgata aaggctatat aatctcaaat gaatgggctg 2040
aggatggtgt tcacaggtgc tttagtgaag tccgctcgtg aagagtcgct gaagtgactg 2100
cagatctgtg acgcagtcgt tttgggcaga cggccgttga aattcggttg agtaattgat 2160
accaggtgag gctagaggat gtagaaattc atttgtgtag aatttaggga gtggccctgg 2220
cgtgatgaat gtcgaaatcc gttccttttt actgaaccct atgtctctgg ctgagtgcca 2280
caccgccggc agccgcaaag cgtctcaaac cattgccttt tatggtaata atgagaatgc 2340
agagggactt cctttgtctg gcacatctga ggcgcgcatt gtcacactag cacccactag 2400
cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca 2460
ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta 2520
cagttacagc catggcgtat cgatgtcgac tttttttttt ttttttacat ggaataaaat 2580
tttaatttat tctcgttggc tacaggggtt tatcttcatc ctttatgctt ttacactctt 2640
cctcgtcttt tgggaaatct cagttctttc agaggcaaac cttcagcatc cacttcattc 2700
actatgtgta tcggtgaaag ttgttccatt ggagagtctg caggaatgga tagtactgca 2760
tcaccagcat ccatcatcct aaatgttgcc tccacttcac caacgcttct ctttccacca 2820
ggaagccaac catatgacca atgctggcaa agccatgggg gagagcagcc tcagagagag 2880
ctttcgtctt ctggcttgct tcaagaagga catgcacaag gtggaaactt acctgagggt 2940
tgcgaattgc aggagatccc tggattcaaa ctgcaccctg tagatggcgc caatgtattg 3000
ctagccaaag cctgtgacac actttgctgc aaatctaaaa ccagtttaag tcctcaaaat 3060
ctcctaatat aattattatc tggtcttata tatgcaggaa atgtcaacca ggcatggcta 3120
ggtctgttct ctagttccct cccatatcta aacccaacac tattgtattt attcttctca 3180
ttggggagtg ctcgtaaatt aaagacatta agatctgatt taacatttca cagtggtgct 3240
aagcaatata tggcaatata ttttcaaatg tgcccaaatc gctttgactc tagtatttta 3300
tggctccaaa aatggctaaa gatgcctttt gtcgaaactg tcatttggat ggatgggttc 3360
actcccaacc aagtgtatga atgtaaacat ttgtctgtct gatagattat gtccatatta 3420
ttagctcatg ctgttctctt gaagctgtgt gtctttatcc attaaatttc taaactgcat 3480
ccaatgtgtc tgctgtatga cctgtgtggt ttattttttt ctacccgtct caacattaca 3540
cgtaccttac attttcactg ctcggatatt catcaaaatc agtcctatca tccgtccaca 3600
atgaacgagt tcaaaatgga ccgaaaaaaa tgaccagcgg aattc 3645
Claims (2)
1、一种不育转基因鱼的制备方法，包括下列步骤：
A、鲑鱼型促性腺激素释放激素反义RNA表达载体的构建，在鲑鱼型促性腺激素释放激素反义RNA表达载体中，以鲤鱼肌动蛋白基因的启动子2.5kb片段为启动子，按3′-5′的方向，286碱基对鲤鱼鲑鱼型促性腺激素释放激素基因的cDNA片段为靶位基因插入在启动子的下游，随后以草鱼生长激素基因的3′侧翼顺序0.8kb为终止顺序，反义载体骨架为pUC118；
B、转基因鱼的制备，将鲑鱼型促性腺激素释放激素反义RNA表达载体分别用限制性内切酶消化，得到线性重组DNA片段，回收的DNA溶于缓冲液，调节其终浓度为50ng/μL，鲤鱼受精卵用0.25％的胰蛋白酶消化去除卵膜，采用显微注射的方法，在第一次卵裂前将DNA溶液导入受精卵动物极，DNA注射剂量约5×105拷贝/卵，完成显微操作后的受精卵置于实验室无菌环境中孵化；
C、不育转基因鱼的筛选，剪取转基因鱼尾鳍，提取DNA，采用PCR技术检测转基因阳性鱼；从转基因阳性鱼尾部血管取血，检测转基因阳性鱼血清中11-酮基睾酮的浓度，确定雄性鲤鱼，同时采用双抗体放射免疫法测定血清的促性腺激素含量，其血清促性腺激素含量低于同批转基因阳性鱼促性腺激素含量的60％，则筛选获得不育的转基因鱼，正向检测引物为5′-CCATGGCGTATCGATGTCGAC-3′位于启动子与反向靶向序列之间的接头序列上，反向检测引物为5′-CATGGCTTTGCCAGCATTGG-3′位于靶向序列上，两引物扩增的片段长度为328bp。
2、根据权利要求1所述的一种不育转基因鱼的制备方法，其特征在于鲑鱼型促性腺激素释放激素反义RNA表达载体的重组基因是：
aagcttttag accttcttac ttttggggat tatataagta ttttctcaat aaatatctca 60
tatcttactg tggtttaact gctgaatcta aaattttaat acaaaagtag ttatatttgt 120
tgtacattgt aaactataac ttaacttcag tttcagagaa actcatgtgc tcaaaatgta 180
aaaaaagttt cctgttaaat attttgtaaa tgtattgaag acaaaataag aaaaaaaaaa 240
tataagccac taaatcacac tgtccttggt atcagcaaga gattctgaca taatcagctg 300
tttttgttta ttactgccat tgaaggccat gtgcattagt cccaagttac acattaaaaa 360
gtcacatgta gcttaccaac atcagtgctg ttcaagcaca gcctcatcta ctattcaaac 420
tgtggcacca tctaaaatat gccagaattt ttttatttaa tgaatttgac cctgaaatat 480
gtattaatat cactcctgtg atttttttgt aatcagctta caattacagg aatgcaagcc 540
tgattcatta caagtttcac tacactttct ctgacaacat cacctactga actcagacca 600
gctagttgct ccttaagtat acaatcatgt cactaatcct catttcaatg aaaaataccc 660
ctattgtact tggtacttgg tagataacca cagagcagta ttatgccatt attgtgaata 720
caataagagg taaatgacct acagagctgc tgctgctgtt gtgttagatt gtaaacacag 780
cacaggatca aggaggtgtc catcactatg accaatacta gcactttgca caggctcttt 840
gaaaggctga aaagagcctt attggcgtta tcacaacaaa atacgcaaat acggaaaaca 900
acgtattgaa cttcgcaaac aaaaaacagc gattttgatg aaaatcgctt agggatcccc 960
ccttgctctt caaacaatcc agcttctcct tctttcactc tcaagttgca agaagcaagt 1020
gtagcaatgt gcacgcgaca gccgggtgtg tgacgctgga ccaatcagag cgcagagctc 1080
cgaaagttta ccttttatgg ctagagccgg gcatctgccg tcatataaaa gagcgcgccc 1140
agcgtctcag cctcactttg agctcctcca cacgcagcta gtgcggaata tcatctgcct 1200
gtaacccatt ctctaaagtc gacaaacccc cccaaaccta aggtgagttg atctttaagc 1260
tagcttttta cattttcagc tcgcatatat caattcgaac gtttaattag aatgtttaaa 1320
taaagccaga ttaaatgatt aggctcagtt accggtcttt tttttctcat ttacgtgcga 1380
actctgctta aactctagtt attctttatt aatatgtggt tatttttata tatgtatgtg 1440
ttatcataac tgtactggct atgtcaggtg gtaatgactg taacgttacg ttactcgttg 1500
taggcacgac attgaatggg ccggtgttga aataagtctt caaccccttt taacctcaaa 1560
atgtgctctg gttaacaagg attttaacag ctatcagtat gactgtgcgg ttttaaagcc 1620
gttagtgagg cacgttgcac acttgatgga tggccggaat gggaagttct ttatgcaggc 1680
agtgctgcag cagggtgtga cctactttag ctaacgttag ccggctaacc agcattcatc 1740
tgccggtaac ttgagtctaa tattctctca tgtgatatcg aagtgatcaa agacacgtct 1800
gttagctcac tttaaccaac tgtagtgaaa aatagcgcag tgtgcagccc ttcagtcttt 1860
catttaggct gattattcaa tcattttatt aactattaac gcgttactaa acgtaaggta 1920
acgtagtcag tttttaataa ctggtgaaaa gtactggttg ggtttaaatg gtgacttata 1980
attgtgttgg agggggaaac ctttttgata aaggctatat aatctcaaat gaatgggctg 2040
aggatggtgt tcacaggtgc tttagtgaag tccgctcgtg aagagtcgct gaagtgactg 2100
cagatctgtg acgcagtcgt tttgggcaga cggccgttga aattcggttg agtaattgat 2160
accaggtgag gctagaggat gtagaaattc atttgtgtag aatttaggga gtggccctgg 2220
cgtgatgaat gtcgaaatcc gttccttttt actgaaccct atgtctctgg ctgagtgcca 2280
caccgccggc agccgcaaag cgtctcaaac cattgccttt tatggtaata atgagaatgc 2340
agagggactt cctttgtctg gcacatctga ggcgcgcatt gtcacactag cacccactag 2400
cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca 2460
ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta 2520
cagttacagc catggcgtat cgatgtcgac tttttttttt ttttttacat ggaataaaat 2580
tttaatttat tctcgttggc tacaggggtt tatcttcatc ctttatgctt ttacactctt 2640
cctcgtcttt tgggaaatct cagttctttc agaggcaaac cttcagcatc cacttcattc 2700
actatgtgta tcggtgaaag ttgttccatt ggagagtctg caggaatgga tagtactgca 2760
tcaccagcat ccatcatcct aaatgttgcc tccacttcac caacgcttct ctttccacca 2820
ggaagccaac catatgacca atgctggcaa agccatgggg gagagcagcc tcagagagag 2880
ctttcgtctt ctggcttgct tcaagaagga catgcacaag gtggaaactt acctgagggt 2940
tgcgaattgc aggagatccc tggattcaaa ctgcaccctg tagatggcgc caatgtattg 3000
ctagccaaag cctgtgacac actttgctgc aaatctaaaa ccagtttaag tcctcaaaat 3060
ctcctaatat aattattatc tggtcttata tatgcaggaa atgtcaacca ggcatggcta 3120
ggtctgttct ctagttccct cccatatcta aacccaacac tattgtattt attcttctca 3180
ttggggagtg ctcgtaaatt aaagacatta agatctgatt taacatttca cagtggtgct 3240
aagcaatata tggcaatata ttttcaaatg tgcccaaatc gctttgactc tagtatttta 3300
tggctccaaa aatggctaaa gatgcctttt gtcgaaactg tcatttggat ggatgggttc 3360
actcccaacc aagtgtatga atgtaaacat ttgtctgtct gatagattat gtccatatta 3420
ttagctcatg ctgttctctt gaagctgtgt gtctttatcc attaaatttc taaactgcat 3480
ccaatgtgtc tgctgtatga cctgtgtggt ttattttttt ctacccgtct caacattaca 3540
cgtaccttac attttcactg ctcggatatt catcaaaatc agtcctatca tccgtccaca 3600
atgaacgagt tcaaaatgga ccgaaaaaaa tgaccagcgg aattc 3645
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| CNB031254551A CN1294808C (zh) | 2003-09-26 | 2003-09-26 | 不育转基因鱼的制备方法 |
| US11/094,286 US7230156B2 (en) | 2003-09-26 | 2005-03-31 | Method for preparing a sterile transgenic carp |
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| CNB031254551A CN1294808C (zh) | 2003-09-26 | 2003-09-26 | 不育转基因鱼的制备方法 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1955738B (zh) * | 2005-10-28 | 2012-01-11 | 中国科学院海洋研究所 | 一种鲆鲽鱼类受精卵微管骨架免疫荧光染色观察方法 |
| CN102363792A (zh) * | 2011-11-11 | 2012-02-29 | 中国水产科学研究院淡水渔业研究中心 | 制备IGF2b转基因鱼的慢病毒方法 |
| CN105764333A (zh) * | 2013-11-15 | 2016-07-13 | 马里兰大学巴尔的摩县 | 生产不育的鱼和产卵水生动物及递送化合物至卵和胚胎中的方法 |
| CN107385070A (zh) * | 2017-08-22 | 2017-11-24 | 山东出入境检验检疫局检验检疫技术中心 | 快速鉴定转基因三文鱼及其制品的方法 |
| CN109874707A (zh) * | 2019-04-08 | 2019-06-14 | 中国科学院水生生物研究所 | 一种高效创制异源八倍体银鲫的方法 |
| CN110050736A (zh) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | 一种侏儒蛤卵膜的溶解方法 |
| US10709119B2 (en) | 2015-05-19 | 2020-07-14 | University Of Maryland Baltimore County | Methods of agent delivery into eggs and embryos of egg-producing aquatic animals for drug screening, agent toxicity assay and production of infertile fish |
| CN113163740A (zh) * | 2018-08-10 | 2021-07-23 | 水产养殖技术中心有限公司 | 一种产生不育和单性后代的方法 |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2253937B2 (es) * | 2003-01-14 | 2007-01-01 | Emilio Espinosa Velazquez | Maquina hiperbarica automatica para triploidizacion de perca y trucha. |
| CN103088066B (zh) * | 2013-02-07 | 2014-01-15 | 中国科学院水生生物研究所 | 一种控制鱼类生殖的方法 |
| CN104396809A (zh) * | 2014-10-18 | 2015-03-11 | 颍上县润海畜禽养殖有限公司 | 一种草鱼的养殖方法 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2191969A1 (en) * | 1995-04-06 | 1996-10-17 | James R. Wright, Jr. | Transgenic fish in the treatment of diabetes |
| US6476290B1 (en) * | 1995-04-19 | 2002-11-05 | Dalhousie University | Transgenic tilapia comprising a humanized insulin gene |
-
2003
- 2003-09-26 CN CNB031254551A patent/CN1294808C/zh not_active Expired - Fee Related
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2005
- 2005-03-31 US US11/094,286 patent/US7230156B2/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1955738B (zh) * | 2005-10-28 | 2012-01-11 | 中国科学院海洋研究所 | 一种鲆鲽鱼类受精卵微管骨架免疫荧光染色观察方法 |
| CN102363792A (zh) * | 2011-11-11 | 2012-02-29 | 中国水产科学研究院淡水渔业研究中心 | 制备IGF2b转基因鱼的慢病毒方法 |
| CN105764333A (zh) * | 2013-11-15 | 2016-07-13 | 马里兰大学巴尔的摩县 | 生产不育的鱼和产卵水生动物及递送化合物至卵和胚胎中的方法 |
| CN105764333B (zh) * | 2013-11-15 | 2021-09-03 | 马里兰大学巴尔的摩县 | 生产不育的鱼和产卵水生动物及递送化合物至卵和胚胎中的方法 |
| US10709119B2 (en) | 2015-05-19 | 2020-07-14 | University Of Maryland Baltimore County | Methods of agent delivery into eggs and embryos of egg-producing aquatic animals for drug screening, agent toxicity assay and production of infertile fish |
| CN107385070A (zh) * | 2017-08-22 | 2017-11-24 | 山东出入境检验检疫局检验检疫技术中心 | 快速鉴定转基因三文鱼及其制品的方法 |
| CN113163740A (zh) * | 2018-08-10 | 2021-07-23 | 水产养殖技术中心有限公司 | 一种产生不育和单性后代的方法 |
| CN113163740B (zh) * | 2018-08-10 | 2023-09-08 | 水产养殖技术中心有限公司 | 一种产生不育和单性后代的方法 |
| CN109874707A (zh) * | 2019-04-08 | 2019-06-14 | 中国科学院水生生物研究所 | 一种高效创制异源八倍体银鲫的方法 |
| CN110050736A (zh) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | 一种侏儒蛤卵膜的溶解方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1294808C (zh) | 2007-01-17 |
| US20050257281A1 (en) | 2005-11-17 |
| US7230156B2 (en) | 2007-06-12 |
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