animal cloning
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ANIMAL CLONINGBLS 308/209HOZA, A.SMAY, 2009
Types of cloning
• Recombinant DNA cloning
• Therapeutic cloning– Stem cells
• Reproductive cloning– Embryo splitting
– Somatic cell nuclear transfer (SCNT)
History of cloning by SCNT
• Nuclear transfer was performed with frogs in the 1950s
• Cattle were cloned by transfer of embryonic nuclei in the 1980s
• Dolly was the first animal cloned by SCNT from an adult cell in 1997
Animals cloned by SCNT
• Sheep, pig, goat, cattle, cat, rabbit, mouse, mule, horse, rat, deer, fish
• Cloning efficiency for Dolly, 1/277 reconstructed embryos (0.3%)
Basic SCNT Methodology
Injection of donor cell under the Zona Pellucida
Injection of donor cell or Isolated nucleus into The cytoplasm
SCNT-chromatin transfer
• Chromatin condensation• Fusion with an enucleated egg
• Enhanced survival of clones
Challenges to successful SCNT
• Reprogram a nucleus from a differentiated stage (somatic cell) to an embryonic stage
• Properly activate genes necessary for early embryonic development and suppress differentiation associated genes
• Dog cloning- Missiplicity Project
• Cat cloning
Shin et al., 2002
Pet cloning
Clones of clones
• Mice have been serially cloned to 6 generations
• Cattle have been serially cloned to 2 generations (3rd generation failed)
Donor
clone 1
clone 2Kubota et al, 2004
Literature survey of developmental problems in cloned animals
77% of clonedAnimals arehealthy
Cibelli, 2002
Embryonic problems with SCNT
• High mortality during gestation
• >65% of one cell cloned embryos fail to develop to morula/blastocyst
• Days 30-60– Cloned cattle: 50-100% embryonic loss– Normal service: 2-10% loss– In vitro fertilization: 16% loss
• Increased incidence of spontaneous abortions during second trimester
• Abnormal placentas
Postnatal problems with SCNT
• Abnormally high birth weight
• Respiratory and metabolic abnormalities– Lung dysmaturity
– Pulmonary hypertension
• “Adult clone sudden death syndrome” -cloned pigs died of heart failure at less than 6 months
Factors contributing to early embryonic death
• Extensive micromanipulation of oocytes andsomatic cells
– Enucleation of an oocyte, removal of 5-15% of ooplasm– Electrical induced fusion
• Incompatibility between mitochondrial and nuclear DNA
• Incomplete reprogramming of somatic nucleus
• Immunological rejection of SCNT fetuses -abnormal MHC-1 expression by trophoblast cells
Early death of cloned mice
Ogonuki et al., 2002
Abnormal expression of genes in cloned mice
• Global analysis of gene expression in cloned mice using DNA microarrays
Humphreys et al., 2002
Cloned mice
• Cloned mice have an obese phenotype
• Phenotype is not transmitted to offspring
• Obesity due to epigenetic changes
Tamashiro et al., 2002
Premature ageing of clones?
• Telomeres are specialized structuresat the ends of linear chromosomes that shorten with age
• Cloned cattle have widely varying telomere lengths
• Dolly’s telomeres– shorter than an age-matched control– consistent with a 6-year old mammarycell
telomere
Advantages to SCNT
• Restore endangered species
• Allows for the targeted deletion (knockout) of genes in farm animals
• Alternative method for generating transgenic animals as bioreactors (pharming)
Cloning of endangered species
• Gaur bull calf cloned using a domestic cow embryo and surrogate mother• Cells from skin cells frozen for 8 years• Clone died 48 hours after birth
Lanza et al., 2000
Cloning of endangered species
• Cloned African wildcat
• Nucleus from frozen cell transferred into domestic cat oocyte
Xenotransplantation
• Transfer of cells, tissues or organs between species
• Pigs contain alpha galactosyl sugars on the cell surface.
• Human and higher primate lack alpha-galactosyl sugars; thus have antibodies to alpha-gal
• Alpha-gal antibodies lead to acute tissue rejection
Gene knockout technology
• Combine SCNT with somatic cell gene knockout technology
Kent-First and First, 2000
Gene knockout in farm animals
• Cloned pigs lacking the alpha-galactosyltransferase gene - xenotransplantation
• Cloned cattle lacking prion gene
Transgenic animal bioreactors
• Produce important human pharmaceutical proteins in milk
• Clones would provide a genetically stable line for protein production
Advantages to cloning transgenic animals as bioreactors
• Increase efficiency to 100% starting with a transgenic cell line
• Can predetermine sex
• Allows for rapid expansion of genetically identical animals
Future cloning issues
• Food consumption risks of cloned animals– Health of animal clones
– Composition of meat and milk
• No differences in milk composition were seen between clones and control dairy cattle
– total solids, fat, lactose, and protein (Norman and Walsh, 2004)
• Many different species have been cloned
• Animal cloning has many successes but also many complications
• Refinement of technique will overcome some of these problems
• Cloning Applications– transgenesis
– gene knockout