CLONING IN FARM ANIMALS

PROBLEMS & PERSPECTIVES

SHAFQUT MAJEED KHANM.V.Sc Scholar

Animal BiotechnologyS.K. University of Agricultural Sciences & Technology

Shuhama, Srinagar, J&K, India, 190001

WHAT IS CLONING?

Cloning is an ambiguous term

Molecular cloning : Duplication of strings of DNA containing genes in a host bacterium

Cellular cloning: Copies of a cell are made, resulting in a ‘cell line’

Embryo twinning: Embryo which has already been formed sexually is split into two identical halves

INTRODUCTION

Greek word ‘klon’ referring to process whereby

a new plant is created from a twig

An organism produced non sexually which genetically resembles the original organism

No clone, theoretically has all the 100% DNA from a single parent

CLONE

Procedure of creating a whole new

multicellular organism which is genetically identical to original organism

Non sexual

No fertilisation or inter-gamete contact takes place

ANIMAL CLONING

1891: Hans Driesch separated the

blastomeres of a two-cell embryo of sea urchin mechanically by shaking them in seawater resulting in two whole sea urchins (Driesch, 1891)

Hans Spemann repeated in a vertebrate (salamander) using a hair from his baby boy to separate the cells

(Spemann, 1902)

HISTORY OF CLONING

Unavailability of efficient handling system

Lack of recognition that mammalian oocytes

and embryos require strictly controlled temperature for

development

No success in cloning

for 80years

Eventually the first successful embryo splitting was

performed in domestic animals with the purpose of rapid multiplication of valuable individuals

(Willadsen, 1979; Ozil et al., 1981) 1981: The first success in mammalian nuclear transfer

The birth of three mice was reported following the trans-plantation of early embryonic cells into enucleated zygotes (one-cell embryos) Illmensee and Hoppe (1981)

CLONING IN MAMMALS

However, nobody (including the authors)

could repeat this experiment

Embryologists declared that the cloning of mammals by simple nuclear transfer was biologically impossible (McGrath and Solter, 1984)

In 1986, cloning technology was successfully established

Cloned sheep were created from embryonic cell nuclei transferred into enucleated oocytes (Willadsen,1986)

Same technique was successfully established in goat, cattle, pig etc.

Embryonic cell donors were always used

EARLY SUCCESS

SCNT is the technique of transferring the nucleus of a

somatic cell into an oocyte and activate it to develop into a full embryo

Technique established by the birth of world’s first cloned mammal from adult somatic cells, Dolly, in 1996 (Wilmut et al., 1996)

Subsequently, cloning by SCNT has been successful in Cattle, Mice, Pigs, Goats, Rabbits etc.

(Edwards et al.,2001)

CLONING BY SCNT

Given the incredible complexity of what must be

involved in the ooplasm redirecting functionality of a somatic nucleus, it is remarkable that any cloned embryos develop at all

Very little is known about specific factors or mechanisms involved with the ooplasm reprogramming a somatic nucleus (Edwards et al., 2010)

REMARKABLE

Collection of Ovaries

In-Vitro Maturation of Oocytes

Enucleation of oocytes

STEPS IN CLONING

Fusion of somatic cell nucleus with recipient oocyte

Activation & Culture Of Embryos

Embryo Transfer into recipient animal

enucleation

reconstructed embryos blastocysts

clones

egg donor

donor cell

Somatic cell donor

recipient cytoplast

foster mother

Cloning Procedure

B I O M E D IC A L

AGRICULTURAL

RESEARCH

Applications of cloning

Cloning at present the most efficient way to

produce transgenic farm animals. By introducing the genetic changes into cells, and

then choosing the ones with the desired changes for the cloning, these animals could be then used as:

BIOMEDICAL

DISEASE MODELS

BIOREACTORS

XENOTRANSPLANTATION

Transgenic animal produtionby cloning

enucleation

reconstructed embryos

blastocystsclones

egg donor

donor cell

Somatic cell donor

recipient cytoplast

foster mothertransgene

Animals designed to express, either at the

genotypic or phenotypic level, a certain human disease

Can be used both to further understanding of the disease and to do initial tests on possible treatments

Sheep & especially pig are more suitable disease models for humans than mice (Gjerris et al., 2007)

DISEASE MODELS

Candidate genes are available for establishing

possible models of human diseases

Neurodegenerative (Parkinson, Alzheimer) diseases, Skin alterations (psoriasis, epidermolysis), Diseases with suspected or known genetic background

(certain types of diabetes mellitus, arteriosclerosis and breast cancer).

(Vajta et al., 2006)

This will be the earliest application of cloning

Bioreactors are transgenic animals that have

had genes that produce human proteins inserted into their genome

Research in this field has been designed to get the transgenic animals to express the desired proteins in their milk

BIOREACTORS

Goats, cows, sheep and pigs have been

genetically modified and cloned in attempts to create such proteins

Proteins such as human coagulation factor IX, Human anti-thrombin and Alfa-1-antitrypsin have all been harvested in an experimental setting from transgenic and cloned animals today

The transplantation of organs between distinct species

Increase of the life expectancy and advancement in transplantation technology has increased exponentially the number of patients waiting for transplantation, while the quantity of available organs is stagnating, or even decreasing

(Niemann et al., 2011) 30% lag between patients & available organs (Davies et al., 2011)

XENOTRANSPLANTATION

Pigs appears to be the most suitable donor

animal

But there is problem of immunological rejection & Hyper-acute rejection (HAR)

Another problem is acute vascular rejection (AVR), which occurs within a few days of transplantation

(Gjerris et al., 2007)

Enzyme 1,3-galactosyltransferase (present on cell

surfaces in almost all mammals with the exception of humans, apes and Old World monkeys) is involved in the HAR response

(Phelps et al., 2003)

Pigs have been produced that have disrupted genes for the enzyme thus being unable to express 1,3-gal sugars on the cell surfaces (so called knock-out pigs) (Lai et al., 2002; Fishman and Patience, 2004)

Cloning can be used to produce stem cells

Stem cells could be further used for various regenerative medicine techniques

Before human stem cell trails, cloning provides an opportunity to first produce stem cells in animals for better understanding

STEM CELL RESEARCH

A possibility that lies further in the future than

biomedical uses

Cloning can be used to create copies of animals with highly valued traits, such as a dairy cow with high milk production or proven bulls

To reserve elite genetic resources (Japanese Black steers of superior beef quality were reserved & recreated).

(Shioya et al., 2006)

AGRICULTURAL APPLICATIONS

Improvements in livestock can also be made by inserting genes of desired qualities into the donor cells prior to nuclear transfer

Improvement of traits such as fast growth rate, leanness of the meat or increasing milk yield could be achieved

Livestock Improvement

Propagation of elite germplasm could be achieved by

developing male clones of a highly valued animal & then further propagating germplasm by AI

Production of a large number of clones from high quality animals (i.e. from within the nucleus herd) which will allow overall genetic improvement of the herd

(Woolliams & Wilmut 1999)

Genetic Improvement

CONSERVATION OF ENDANGERED SPECIES

Simple Cloning

• Creating clones from somatic cells of endangered species

• For recovering rare domestic breeds

• Enderby Island Cattle being cloned currently

• Many threatened Indian livestock could be recovered

Cross Species Nuclear Transfer

•For recovering endangered wild animals•Somatic cell nucleus transferred into oocyte of a closely related domesticated species•Hangul & Cheru could be recovered by Cross species cloning

Clones provide unique experimental material since they are genetically identical

Especially useful in drug & feed trails

RESEARCH APPLICATIONS

Cloning provides insight into natural

reproductive development

Helps to better understand the biology of zygotes, embryos & embryonic development & anomalies.

This could be further utilised for both human & animal welfare

Understanding Embryology

CRITICISM

•DEVELOPMENTAL ANOMALIES

•SHORT LIFE SPAN

•LARGE OFFSPRING SYNDROME

•AGEING OF CLONED ANIMALS

•SAFETY APPREHENSIONS

PROBLEMS

Low pregnancy

rates

High level

of losses during early and late

pregnancy

Stillbirths

Early postnatal

deaths

DEVELOPMENTAL ANOMALIES

These all depend upon technical expertise and can be sorted out and optimised in future

Causes•Inappropriate cell cycle phase synchrony

• Inappropriate handling of oocytes, somatic cells and embryos

• Various manipulations and cultural techniques

Myth spread due to early death of Dolly

Dolly died of retroviral Disease (Bridget et al., 2003)

Almost all the cloned animals have a normal life span

(Trounson et al., 2007)

Short Life Span?

Most Common in Cattle & Sheep

Not observed in goats & pigs (Betthauser et al., 2000;Reggio et al., 2001;Behboodi et al., 2005)

LOS involves large sized offspring, abnormally overweight, and large visceral organs & oversized umblicus

Large Offspring Syndrome

Abnormal hypomethylation status at an

imprinting control region of Kcnq1ot1/Cdkn1c domain in cattle

Altered expression of IGF2 & IGFBP2 in sheep

Insulin-like growth factor (IGF) system plays a major role in regulating growth of the fetal sheep (Wilmut et al, 2000)

Possible causes?

LOS is not seen in cloned animals only (Young et al.,

1999) Not all the LOS offsprings die

Loss is still manageable as compared to profit

Better understanding of epigenetic mechanism will help possibly eliminating it in future

(Vajta et al., 2006)

Justification

ARE CLONED ANIMALS ALREADY AGED???

AGEING

There is telomere shortening after each cell

cycle which possibly works as biological clock (Xu and Yang, 2003) Telomere shortening in the somatic cells to be

used for cloning would already have occurred

The telomeres of Dolly were also found to be relatively shorter (Shiels et al., 1999)

Length of the telomeres depends on not just

the age of the donor animal but also a range of parameters, including cell type, cell culture, nuclear transfer procedure, sampling and the measuring protocol

(Xu and Yang, 2004)

Telomere shortening was not confirmed in subsequent findings (Vajta et al., 2006)

Justification

About human consumption of products from cloned animals

No biological & nutritional difference between milk & milk products of normal & cloned animals

(Tome’ et al., 2004) No significant difference between meat of normal &

cloned animals (Walker., 2006)

Approved by FDA, Dec,26,2006

SAFETY APPREHENSIONS

Cloning is a developing research field & active efforts for farm animal cloning are going on in more than 30 countries including:

New Zealand & Australia UK & other European countries U.S.A. & Argentina China, Iran & India etc.

Support is provided to scientists to explore cloning & utilise all its potential benefits

CURRENT SCENARIO

At NDRI Karnal & SKUAST Kashmir

Worlds first cloned buffalo & pashmina goat produced here

Success rates still very low

Research going on to improve efficiency & applicability of animal cloning

In INDIA

Cloning is a fast developing technique, far from

being optimized

Evolution in the efficiency and applicability in new species is expected

Basic research in nuclear reprogramming will continue, especially due to connections to the embryonic stem cell research field.

PERSPECTIVES

Practical application of nuclear transfer in the near future is expected in the agriculture for enhanced food production and propagation of superior breeding stock

In medicine by using animals as novel disease models, bioreactors and organ donors produced by the combination of transgenesis/homologous recombination in somatic cell lines and nuclear transfer; and in therapeutic cloning of human embryos

Methods with much improved efficiency would provide a good tool for endangered species preservation efforts, as well

Further research is needed to improve success

rate & remove the anomalies & apprehensions related to cloning

Thank You