公开(公告)号：US20120017289A1

公开(公告)日：2012-01-19

申请号：US12895913

申请日：2010-10-01

申请人： Subeer Suhash Majumdar ,  Deepika Sharma ,  Neerja Wadhwa

发明人： Subeer Suhash Majumdar ,  Deepika Sharma ,  Neerja Wadhwa

IPC分类号： C12N15/85

CPC分类号： A01K67/0275 ,  A01K2207/05 ,  A01K2227/105

摘要： The present invention provides a novel method of generating knock down models by electroporation of shRNA construct into the testis. The present invention provides an ethically superior, non-surgical, user friendly rapid method for the generation of permanent lines of shRNA knock down non human vertebrates. This invention is ethically superior as it does not involve any loss of animal life and drastically minimizes the production time and use of animals. Current techniques for making knockout models are cumbersome, require trained personnel, costly infrastructure and require hundreds of eggs collected after killing several females. In contrast, this method neither involves any costly infrastructure nor requires trained personnel. The invention also relates to the quick incorporation of shRNA gene construct into the germline of a species so that shRNA is inheritable. The present invention also generates in a single go a variety of knock down models differentially expressing gene specific shRNA, depending on differential shRNA gene incorporation in native genome of various male germ cells, so that there is no restriction in the choice of the gene knock down.

摘要翻译： 本发明提供了一种通过将shRNA构建体电穿孔进入睾丸来产生敲除模型的新方法。 本发明提供了一种伦理上优于非手术的，用户友好的快速方法，用于产生shRNA敲除非人脊椎动物的永久性线。 本发明在伦理上是优越的，因为它不涉及动物生命的任何损失，并且极大地最小化了动物的生产时间和使用。 目前用于制作敲除模型的技术很麻烦，需要经过培训的人员，昂贵的基础设施，并且在杀死几名女性后需要收集数百个鸡蛋。 相比之下，这种方法既不涉及昂贵的基础设施，也不需要经过培训的人员。 本发明还涉及将shRNA基因构建体快速并入种的种系，以使shRNA是可遗传的。 本发明还单独地产生差异表达基因特异性shRNA的各种敲低模型，这取决于不同的shRNA基因在各种雄性生殖细胞的天然基因组中的掺入，使得在基因敲除的选择上没有限制 。

公开(公告)号：US20120284809A1

公开(公告)日：2012-11-08

申请号：US13102682

申请日：2011-05-06

申请人： Subeer S. Majumdar ,  Deepika Sharma ,  Neerja Wadhwa

发明人： Subeer S. Majumdar ,  Deepika Sharma ,  Neerja Wadhwa

IPC分类号： A01K67/027

CPC分类号： A01K67/0276 ,  A01K2207/05 ,  A01K2267/03

摘要： The present invention relates the function of Dickkopf 3 (Dkk3 ) in testis using shRNA mediated knock down model. Specifically, the present invention provides knock down model comprising reduction in Dkk3 activity. The knock down model of Dkk3 consisting of non human vertebrates which have, incorporated in their genome, shRNA construct targeting mammalian Dkk3 gene exhibits low testis weight, low sperm count and has low litter size. The knock down model displays disrupted seminiferous tubules and are subfertile. The present invention model has a role in sex determination as its interruption leads to sex reversal of XY gonads, converting males to females. Sex reversal role of Dkk3 knock down model can find its utility in agricultural applications. The present invention describes Dkk3 as a dual function protein which is associated with sex determination as well as is essential for the process of spermatogenesis. Such testicular functional studies are useful as a model for various disease states of infertility or subfertility and for identifying a potential treatment to overcome idiopathic infertility.

摘要翻译： 本发明涉及使用shRNA介导的敲低模型的Dickkopf 3（Dkk3）在睾丸中的功能。 具体地说，本发明提供了降低Dkk3活性的敲除模型。 由非人类脊椎动物组成的Dkk3的敲低模型，其在其基因组中并入针对哺乳动物Dkk3基因的shRNA构建体，显示出低睾丸重量，低精子数，并且具有低窝仔数。 击倒模型显示破碎的生精小管，并且是不孕的。 本发明模型在性别决定中起作用，因为其中断导致XY性腺逆转，将雄性转化为雌性。 Dkk3敲击模式的性逆转作用可以在农业应用中发现其效用。 本发明将Dkk3描述为与性别决定相关的双功能蛋白质，并且对于精子发生过程是必需的。 这种睾丸功能研究可用作各种不孕症或不孕症状态的模型，并用于鉴定克服特发性不孕症的潜在治疗。