After Megan & Morag:An overview of animal cloning and embryonic stem cells

Jyh-Cherng Ju (朱志成),Professor

Department of Animal Science,National Chung Hsing University,Taichung, Taiwan.

May 5, 2009

Megan & Morag (born June 1995) were the first cloned mammals from cultured differentiated cells.

The ewe 2, U-2 or you too ?

The production of Megan & Moragdemonstrated that viable sheep can be produced from cells cultured in vitro.

Dr. Keith CampbellEmbryo-derived fibroblasts (TNT4)(Passage 6, Welsh mountain sheep embryos)

They signified the technical break-through that made Dolly the sheep possible.

The birth of Megan and Morag, [with their huge beneficial potential] made no headlines.

“Few celebrities have so besotted the world’s media as she did.”

“She posed for People magazine, became a cover girl, and even caught the eye of Bill Clinton.”

She was survived by five of her lambs.

Dolly was cloned from the frozen mammary cells of an adult sheep and born on 5th July, 1996.

Arthritis and progressive lung disease

Dolly was put into sleep on 14th Feb, 2003.

The ability to clone a mammal was the culmination of research in cell and developmental biology since 1800s.

New Biology (1970s):The cell/Animal cloningStem cell research/Gene therapyCancer/Aging

Agar chips

Blackface oviducts

29 blastocysts(removed from the agar chips)

How was Dolly cloned?

Ovine mammaryEpithelia (OME)

ET to 13 Blackfacesrecipients

1/277 became Dolly

Dolly & Bonnie

X Welsh mountain ram (David)

1998

2000

2001

Dolly and Bonnie

Megan and MoragBorn dead or died after birth

Low MPFHigh MPFDecreasing (high) MPF Cloning from

cultured embryonic cells

Dolly was produced by GOAT procedure(no adequate time for reprogramming)

NTFusion4-6 hactivation

Fusion+with EP

EP4-6 hNT

The birth of Dolly was announced in Feb 1997:

The 10th Anniversary of the Dolly Experiment.

Publications relating to cloning in mammals (excluding rodents),birds and fish.These data are based on publications identified in Article First on Firstsearch and Web of Science. The search was restricted to English language publications and ignored cloned mice. Although no one database is exhaustive in its coverage, and although we do not believe there is full representation of the scientific literature from the Far East, notably China, we believe the data are sufficient to provide an overview of the field.

Nat Biotech 25:47-53 (2007)

When Dolly was cloned

Geographical distribution of publications

Cloning will not recreate a loved pet

A clone might be 99.95% genetically identical to the original, but it will grow up with a personality and behavior all of its own.

(Harry Griffin)

If the environments are not the same, they are not going to act the same.

Timeline of key events in mammalian cloning.Nat Biotech 25:47-53 (2007)

Dolly takes center stage. Since Dolly was created, interest in cloning mammals has grown.

Grey wolves

African wildcatMouflon2001

2003

Red deer cloned from antler stem cells and their differentiated progeny.(Berg et al., 2007; Biol Reprod 77:384-394)

The significance of donor cell differentiation status was tested:Male antlerogenic periosteum (AP, undifferentiation)Bone cells (induced osteogenesis)Fat cells (induced adipogenesis)

No differences were detected.

Photos from South Korea's Ministry of Science and Technologyshow cats with a gene for producing red fluorescence protein. The cats appear normal in visible light, at left, but their skinglows red under ultraviolet light, at right.

Normal cat(green)

Cloned cat(red)

Cloning dogs is notoriously difficult:

The first cloned dogSnuppy, whose name stands for Seoul National University puppy, was made from a cell taken from the ear of a three-year-old male Afghan hound.

Once growing, it was transferred to Snuppy's surrogate mother, a yellow labrador. The Afghan pup was born by caesarean section after a full 60 days of pregnancy.

the South Korean team only obtained three pregnancies from more than 1,000 embryo transfers into 123 recipients.

Missy’s clones were born on Dec 5, 2007, Feb 15, 2008, and Feb 19, 2008 (left to right).

Missy, the somatic cell donor

Scientists claim to have cloned glowing dogsSouth Korean researchers have named all four beagles ‘Ruppy’

The photo at left shows one of the cloned transgenic dogs named Ruppy, as seen under normal lighting conditions. The photo at right shows the dog under ultraviolet light, glowing red due to the influence of fluorescent genes. Both photos were released by Seoul National University.

The world's first transgenic dogs containing a fluorescent Protein, which make them glow red under ultraviolet light,four months after their birth.

Why/Stem cells in companion animals

Mouse models may not faithfully represent the human disease.

A longer term experiment can be performed.

Similar in size to a neonate or small child.

Physiological parameters (immune system) are closer to those in humans.

(Why/Stem cells in companion animals)

Live under a similar environment as that in humans.

Chronic organ failures, cancer and injuries are severe threats for companion animals.Less ethical barriers to apply new therapies in companion animals.

High economic potential

First cloned female camel born in Dubai. Six-day-old, one-humped female born on Apr 8, 2009.

DUBAI, United Arab Emirates –A scientist says the world's first cloned camel has been produced in the desert emirate of Dubai.

Nisar Ahmad Wani, a senior reproductive biologist at the government's Camel Reproduction Center, says the cloned camel is a six-day-old, one-humped female called Achievement or Injaz in Arabic.

Injaz was born April 8 after an uncomplicated gestation of 378 days, the center said in a press release Tuesday.

(Earlier attempts cloning animals for several generations has been problematic.)

Fourth-generation cloned pigs, developed by Meiji University professor Hiroshi Nagashima, seen in this photo taken onJuly 27, 2007.

The subject to be cloned determines the relative importance of various problems and questions related to cloning. (Solter, 1998, Nature 394: 315-316)

Animals may be benefited by the cloning technology earlier than human beings.

Conservation of endangered species?

“Frozen zoo”

African bongo antelope(600)

Bucardo (0)Spanish Mt. goat

Cheetah (12000)S. Africa

OcelotGaur (Bos gaurus),Habitat: India, Indochina and southeast Asia

Giant panda

Why clone wildlife?

• Cloning is the insurance for conservation failure.

• Cloning may minimize loss of genetic diversity.

• Cloning may rescue already lost genetic diversity.

• Cloning may increase the speed of conservation.

• The speed of breeding program is constrained by the natural limits.

• Breeding programs need large space to keep wildlife stocks. It requires more funding to support the larger number of animals.

Donor cells: skin cells of a male gaur

692 enucleated cow oocytes81 blastocysts (~100 cells/embryo)42 blastocysts/32cows8 pregnancies

2 for scientific analysis4 early abortions (spontaneous)1 late abortion (7 months) 1 gaur was born (Noah)

Snuwolf and Snuwolffy were born October 18 and 26 2005, said Dr Lee Byeong-Chun, a veterinary professor at Seoul National University.

Snuwolf and Snuwolffy, two supposedly cloned wolves at Seoul Zoo.

A paper on cloning wolves contains a factual errorabout earlier research.

This error understated the past success rate and made the apparent technical progress the team had made since then more impressive.

Lee says the mistake was inadvertent and he has asked the journal in question to correct the figure. There are also suspicions that the research team gave incorrect details in a table analysing the mitochondrial DNA sequence of the cloned wolves and their surrogate mother dogs.

But the panel found that the team didnot write a laboratory note when they carried out the research in 2005.

"Materials and documents related to the sampling and analysing of samples were not kept well.

It was also found that the team lacked the ability to systematically analyze the research results“, the panel said in a statement.

We’re about to find out. Researchers in South Korea are getting ready to breed one of the world’s first cloned wolves with a male wolf clone, to test whether the genetically-engineered animals can successfully mate.

Given the precedent set by Snuppy and his mates, Lee and his team suspect that Snuwolffy and her partner won’t have any problemsproducing a litter of Snuwolffy-ettes.

What happens when one cloned wolf mates with another cloned wolf?

We’re about to find out. Researchers in South Korea are getting ready to breed one of the world’s first cloned wolves with a male wolf clone, to test whether the genetically-engineered animals can successfully mate

Seoul National University researcher Lee Byeong-Chun is overseeing the planned tryst between Snuwolffy (maybe it sounds better in Korean?), born in October 2005, and a male clone nearly a year her junior. Lee and his team are no neophytes when it comes to cloning: They produced the world’s first cloned dog, an Afghan named Snuppy (we refrain from judgment on the name), in 2005. Not stopping there, Snuppy then became a father, leading to the first successful breeding of two cloned dogs.

Given the precedent set by Snuppy and his mates, Lee and his team suspect that Snuwolffy and her partner won’t have any problemsproducing a litter of Snuwolffy-ettes.