Mammalian leukocytes contain all the genetic information necessary for the development of a new individual.
We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.
Galli
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Laboratorio di Tecnologie della Riproduzione, CIZ srl, 26100 Cermona, Italy. cegalli@tin.it
Duchi
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Moor
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RM
Lazzari
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D000818:Animals; D001755:Blastocyst; D002417:Cattle; D002459:Cell Fusion; D002875:Chromosomes; D019976:Cloning, Organism / Q000662:veterinary*; D015925:Cryopreservation; D004247:DNA / Q000032:analysis*; D005260:Female; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000502:physiology*; D008297:Male; D008845:Microinjections; D018895:Microsatellite Repeats; D009028:Morula / Q000166:cytology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D010053:Ovary / Q000166:cytology; D011247:Pregnancy
D004247:DNA