Mammalian transgenesis by intracytoplasmic sperm injection

A technology of mammals and sperm, applied in the direction of plant genetic improvement, genetic engineering, micro-injection, etc., can solve the problem of unreliable ability of transgenic animals

Inactive Publication Date: 2002-02-06
UNIV OF HAWAII
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although sperm-mediated DNA transfer to progeny has the potential to significantly simplify the generation of transgenic animals, there is much debate about the efficiency of the live sperm approach to facilitate transgenesis due to its unreliable ability to consistently generate transgenic animals [M. Lavitrano, et al. , 1989, supra; R.N.Brinster, et al., Cell 59, 239(1989); B.Maione, et al., Mol.Reprod.Dev.50, 406(1998)]

Method used

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  • Mammalian transgenesis by intracytoplasmic sperm injection
  • Mammalian transgenesis by intracytoplasmic sperm injection
  • Mammalian transgenesis by intracytoplasmic sperm injection

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0020] Preparation of Fresh Sperm

[0021] Fresh spermatozoa from invertebrates and vertebrates can be collected by methods known to those skilled in the art. For example, mature sperm of rodents (such as mice, golden (Syrian) hamsters, guinea pigs), rabbits, etc. can be collected from the cauda epididymis; Here, mature spermatozoa can be isolated from the freshly ejaculated semen of fertile males. Sperm from fish (eg swordtail, Xiphophorus helleri) and invertebrates such as sea urchin (Tripneustes gratilla) can be collected from the testes of mature males.

[0022] Below is an example of a method for obtaining sperm from the cauda epididymis. The caudate epididymis of mature male mice (approximately 8 weeks or older after birth) was removed. Remove blood and adipose tissue from the surface of the cauda epididymis. It is then compressed, releasing a dense ball of sperm. Place 1 drop (about 2 microliters) of sperm pellet at the bottom of a 1.5ml polypropylene centrifuge tu...

Embodiment 1

[0090] Oocyte Preparation

[0091] Maturation of B6D2F1(C57BL / 6×DBA / 2) was induced by sequential (48 h apart) injections of 7.5 international units (IU) of pregnant mare serum gonadotropin and 7.5 IU of human chorionic gonadotropin (hCG) Female mice superovulate. 14 hours after hCG injection, cumulus-oocyte complexes were collected from oviducts and treated with Hepes-CZB medium supplemented with bovine testicular hyaluronidase (300 USP units / mL, ICN Biochemicals, Costa Mesa, CA) for 3 min , to disperse the cumulus cells. Before injection of sperm nuclei, oocytes were washed and incubated in 5% (v / v) CO 2 Store in CZB medium under mineral oil equilibrated at 37°C in air for up to 4 hours.

Embodiment 2

[0093] Preparation of Fresh Sperm

[0094] Freshly collected sperm from the cauda epididymis of B6D2F1 male mice. Squeeze each epididymis with a finger while piercing its distal portion with sharp forceps. Transfer the dense semen mass exuded from the epididymis to a Petri dish. A few drops (approximately 2 microliters) of sperm were placed at the bottom of a 1.5 milliliter polypropylene microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) and overlaid with 0.2-0.5 milliliters of CZB medium. After approximately 20 minutes of incubation, the upper 0.4 ml of medium was collected and examined. More than 90% of the sperm in this suspension (approximately 3-10×10 6 ) can swim lively.

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Abstract

Co-injection of sperm heads and exogenous nucleic acid encoding the transgene into unfertilized mouse oocytes resulted in embryos expressing the transgene, reflecting the conjugation of the nucleic acid and sperm heads prior to co-injection. Non-selective transfer of coinjected embryos into surrogate mothers yielded offspring expressing the integrated transgene.

Description

[0001] This application claims priority to US Provisional Patent Applications 60 / 096,078 (filed August 11, 1998) and 60 / 133,970 (filed May 13, 1999). [0002] The US Government has a paid license in this invention and has the right under certain circumstances to require the patent owner to license others on reasonable terms under the terms of National Institute of Child Health and Human Development Grant Contract No. HD-34362. Background of the invention [0003] Transgenic animals are important for scientific, pharmaceutical and agricultural uses. The use of genetically engineered livestock to produce foreign proteins in milk is considered a suitable system for the production of therapeutic recombinant proteins. In addition, inserting human genes into the genomes of animals such as pigs allows these animals to be used as living organ or cell "factories" for the production of human organs or cells that will not be rejected by the human immune system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027A01K67/033C12N5/10C12N15/09C12N15/87C12N15/89
CPCC12N15/89C12N15/87A01K2217/05A01K67/0275A01K67/0338A01K67/0335
Inventor R·扬吉马奇
Owner UNIV OF HAWAII
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