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