CLONING:RESULTS MAY VARY

Rainbow and her clone, CC, from TAMU

TEKS Ag: 130.2.12.D 130.3.6.A-D

130.7.6.B 130.7.12.A

From One to Two to Many

What is cloning? DNA Cloning

Transferring DNA into a host cell for recombinant DNA molecule

Reproductive Cloning Generating a new animal with identical nuclear

DNA Therapeutic Cloning aka “Embryo Cloning”

Producing human embryos for research

DNA Cloning

Reproductive Cloning

Form of Asexual Reproduction

Therapeutic Cloning

DNA Collection

Semen Collection Assess potential fertility of male Short or long term storage before AI

Ovum Collection Ovarian hyperstimulation – pharmacologically

stimulated to mature follicles Embryo Collection

Superovulation to release multiple oocytes that can be inseminated

Semen Collection

Performed using artificial vagina, dummy mounting or electroejaculation

Used fresh on the same day, stored for up to 2 days at refrigerator temperature, or frozen for long periods of time

Frozen semen mixed with diluter and extender

Determine concentration and motility Used for AI or IVF

Artificial Insemination

Intracervical Intrauterine Intrauterine tuboperitoneal Intratubal

Artificial Insemination (AI)

PROS CONS

Inseminate more females Avoid injury or physical

breeding problems Control paternity Use semen from males

separated from females by time or distance

Decrease costs of keeping male

Increase revenue of offspring

Increase reproductive success

Decrease contamination and disease transmission

Human Error Some equine breeding

prohibits AI Requires specific

facilities and skilled labor

Increases costs of breeding

Artificial Insemination in the US

Less than 10% of beef cattle; more than 90% of dairy cattle

More than 80% of the swine industry 0% of the chicken industry 100% of the turkey industry

Selectively bred turkey increased the breast size so male turkeys (Toms) are physically incapable of mating

Egg Harvesting Superovulation by injection of Equine

Chorionic Gonadotropin and prostaglandin F2 alpha or by injection of Follicle Stimulating Hormone

Egg can be used for in vitro fertilization Egg can be used for cloning

Genetic material is removed to make an enucleated cell

Somatic Cell Nuclear Transfer

Creating a clone embryo with a donor nucleus Somatic Cell – any cell in the organism that is

not a gamete Somatic cell from adult organism is

collected and the nucleus is transferred into the host enucleated egg

Host cell reprograms donor nucleus and divides mitotically to blastocyst ( about 5 days, about 100 cells)

Cloning

1. Oocytes collected in stage of Meiosis II2. Chromosomes of oocytes removed by

micromanipulation to create cytoplast3. Diploid 2N somatic cells from donor

tissue sample in G0 or G1 of Mitosis4. Somatic cell is combined with cytoplast

by direct injection or by fusion using electric pulse

5. Recombined ooctye artificially activated6. Ooctye begins embryonic development

and is transferred to surrogate

Embryo Collection

Multiple ovum after hormonal ovary stimulation are inseminated, typically by AI

Embryos are non-surgically collected by flushing with a collection line and caught in a filter

Non-surgical Embryo Transfer: embryos packaged into straws and implanted in surrogate dam midway up the uterine horn

Surgical Embryo transfer: embryos are transferred with laparoscopy by penetration of abdominal cavity

Embryo transfer:Fresh: approx. 65% conception rateFrozen: approx. 40% conception rate

Embryo Transfer

Holstein Donor cow with calves carried by beef cattle surrogate dams

Embryo Splitting or Twinning

After fertilization, at the 6 to 8 cell stage, the embryo is split into two

These are used for embryo transfer Both embryos then continue to develop

into two identical twins

Reproductive Cloning

PRO CON

Acquiring better genetic traits in offspring

Producing multiple high quality offspring

Increasing desired traits in a population

Offspring traits are pre-determined

Preserve endangered or extinct species

Decreases genetic diversity

Increased costs of breeding

Unresolved ethical issues

Increased chance of birth defects

Does not ensure exact replica

Famous Clones

CC, 1st cloned cat, 2001

Idaho Gem, 1st cloned equine, 2003

Dolly, 1st cloned mammal, 1996

Snuppy, 1st cloned dog, 2005

Millie, 1st cloned cow, 2000

Freezing Semen and Embryos

Semen extended with glycerol can be frozen with liquid nitrogen (320 degrees below 0)

Frozen semen is stored in straws which can be thawed to directly inseminate a female

Embryo cryopreservation stores pre-implantation stage embryos using slow cooling technique

Frozen embryos in ethylene glycol can be thawed for direct transfer

Frozen embryos in 10% glycerol must be thawed and rinsed multiple times before transfer

Cryogenics

Cryogenics is the engineering of tools to keep very low temperatures

Cryonics is the use of cryogenics in medicnal cryopreservation Frozen semen and embryos for AI and embryo

transfer

Current and Emerging Technologies

Synchronization breeding – using hormones to sync up females coming into heat; typically used for AI or embryo transfer

Superovulation to promote twinning Automatic milking systems – electronic

identification of dairy cattle allows sorting of cows for milking and breeding

Sex Sorting Semen

Selecting semen: beef industry prefers male calves while dairy industry prefers female calves

Controlled Internal Drug Release

Intravaginal progesterone insert to synchronize estrus

Helps to improve results of AI and estrus synchronization

Used in combination with gonadotropin releasing hormone and prostaglandin F2 alpha

Removes need to detect heat Increases pregnancy rate

CIDR