Cloning of an Endangered Species Bos Gaurus: Using Interspecies Nuclear Transfer

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Despite efforts to maintain wildlife conservation as well as biodiversity, many endangered animals are coming closer to the brink of extinction. The current method of preserving genetic diversity in endangered species is to keep them in containments such as zoos. Unfortunately this leads to animals being kept out of their natural habitats, in limited spaces, and causing issues with breeding as well as reproduction failure due to unnatural circumstances created by captivity and potential stress.

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The advances in assisted reproductive techniques allow for other ways to breed endangered species. Unlike domestic animals and their abundant supply of oocytes and surrogate animals, endangered species will require alternative ways of cloning known as interspecies nuclear transfer. This lab report states that interspecies nuclear transfer can be used to successfully clone an endangered species with normal phenotypic as well as karyotypic development by means of development through attachment and the later stages of fetal growth even with the mitochondrial DNA being from another species.

The materials and methods began with extracting skin cells from a deceased male gaur(Bos gaurus). A skin biopsy was minced and cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal calf serum, L-glutamine, nonessential amino acids, mercaptoethanol, and antibiotics at 38 degrees celsius in a humidified atmosphere of 5% CO2 and 95% air(Cibell, Diaz, & Lanza,2000). The tissues were cultivated and a fibroblast cell one layer thick formed. The cell strain was then cryopreserved.

Then bovine (Bos taurus) oocytes were taken from a female cow’s ovaries. Oocytes were amplified at 18-22 hours post maturation. A suspension of actively dividing gaur cells was prepared immediately prior to nuclear transfer. The cell suspension was centrifuged at 800g and 5??L of the resulting cell pellet used for the donor cells(Lanza, 2000). A single cell was placed into the perivitelline space in the fertilized ovum. Cleavage rates were recorded and development to the blastocyst stage was assessed on days 7 and 8 or culture(Lanza, 2000).

Then three fetus’ were sacrificed to be collected by means of C-section at 46 days and 54 days gestation. The fetuses were placed in individual sterile containers and sent to the laboratory to be examined for any abnormalities. The left front leg from each Gaur fetus was removed, minced, and cultured. After 5-10 days, confluent fetal fibroblast cells lines were derived. Cell strains were either subjected to microsatellite marker and cytogenetic analyses, or cryopreserved for long-term storage(Lanza, 2000).

During cytogenetic analysis, cells were treated with colcemid for 20 minutes at 37 degrees celsius in an atmosphere of 95% air and 5% CO2. Cells were then trypsinized and centrifuged for 5 minutes at 200g and the supernatant was removed.

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