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

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. 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. The cells were then re-suspended in a pre warmed hypotonic solution and incubated at 73 degrees celcius for 12 minutes. Cell were then centrifuged and the resulting pellet resuspended in 8mL of Carnoy's fixative at room temperature for 30 minutes. Fixed cells were centrifuged and washed twice in fresh Carnoy's fixative. After the last centrifugation, the cells were resuspended in 0.5 to 1.0mL of freshly prepared fixative and single drops were placed on clear slides and air-dried(Lanza, 2000). Ten Giemsa-banded cells in metaphase arrangement were inspected for complete chromosome numbers for each line. Scientists took photos of five cells in metaphase and one karyotype composed and chromosomes arranged in pairs from largest to smallest.

Mitochondrial DNA was then analyzed using two methods. First restriction fragment polymorphism begins with 0.25??g of total DNA being extracted from different tissues by standard procedures. Oligonucleotides sequences corresponding to positions 16021-16043 and 165-143 of the Bos taurus mitochondrial genome. Although there is a variation in the nucleotide sequence between the Bos taurus and Bos gaurus mtDNA D-loop regions, the oligonucleotide primers have 100% homology with mtDNA from both species. The amplified fragment was labeled with [^32P]-dCTP in the last cycle of the polymerase chain reaction to avoid the detection of heteroduplexes(Lanza, 2000). The second method to analyze the mitochondrial DNA was Allele specific polymerase chain reaction. Oligonucleotide primers corresponding to relatively divergent regions of mtDNA D-loop were used to amplify a 480 bp fragment specifically from taurus or gaurus(Lanza, 2000). The gaurus primers were as follows: forward-CATAGTACATGAACTCATTAATCG and reverse-TTGACTGTAATGCCCATGCC. The taurus primers were as follows: forward-CATAATACATATAATTATTGACTG and reverse-TTGACTGTAATGTCCATGCT. Microsatellite analysis of the bovine chromosome 21 was performed by PCR amplification of the marker after end-labeling one of the oligonucleotide primers with [^32P]-ATP. The amplification products were separated in a denaturing PAGE and analyzed by phosphorimaging(Lanza, 2000).

The results of the cloning experiment with nuclear transfer indicated that at the time the three fetuses were removed, there was no evidence any external abnormalities including any defects in any of the fetuses. Normal development proceeded successfully during the experiment. Everything from the eyes, nose, ears, head, tail, and limb buds were present in fetus number 1. Both fetus numbers 2 and 3 included proportional limbs, with two digits at the distal end including dew claws. Fibroblast cells strains were derived from the cloned animals and subjected to microsatellite marker and cytogenetic analyses(Lanza, 2000). The domestic cattle(Bovidae) have a normal diploid chromosome number of 60. The Gaur has complement of 58 chromosomes. Analysis indicated that the cloned cell strains were normal karyotype of with a complement of 58 chromosomes. The cells observed in each fetus all were in the modal number(89%-92%). After observing the bovine chromosome 21 microsatellite analysis confirmed that all three fetuses had gaurus nuclear background. 11 tissues types were tested and were exclusively taurus. No gaurus mitochondrial DNA was detected in tissues from any of the three fetuses. One fetus and a partial placental unit were recovered after a late term abortion at 202 days of gestation(Lanza, 2000). Body weight was recorded and the male fetus was examined for overall morphology and the skeletal and internal organs appeared to be normal. The fetus appeared to be following normal development and the failure of the pregnancy was likely due to placentation(Lanze, 2000).

Cloning of an animal with the nuclear DNA of one species and the mitochondrial DNA of another species had never been previously reported before this experiment. Since gaurus mitochondrial DNA was not controlled or augmented in the fetuses proposes that sequence variations between taurus(cattle) and gaurus(Gaur) mitochondrial DNA are comparably neutral at the functional level and the fusion product would act as a fertilized egg, eliminating the external mitochondrial DNA. A segment of the highly polymorphic D-loop region of mtDNA is 85% identical between taurus and gaurus, and the nucleotide sequences of the genes for cytochrome b and cytochrome oxidase subunit II are 93% and 94% identical(Lanza, 2000).

In conclusion, this study provides enough detailed evidence that fetal mammals can be generated using interspecies nuclear transfer. Although the cloned fetuses are authentic nuclear (Gaur) clones, they are in fact genetic chimeras with oocyte-derived mtDNA(Lanza, 2000). Since mitochondrial DNA is conveyed by maternal inheritance scientists can predict that breeding of any male offspring would lead to a genetically pure animal. Reverse cloning is also a possibility to develop cows with gaur mitochondrial DNA. The capability to create successful cross species by nuclear transfer would create a new strategy in order to help conservation of species. This process proves to be beneficial in helping endangered species. This study also identifies the importance of preserving and archiving cells for future potential cloning and preservation of species on the brink of extinction.

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