Embryo Splitting and Cloning

Question 1

Fertilization

Answer 1

The fusion of hapliod gametes to form a dipliod zygote

Occurs in the ampulla of the fallopian tube

Question 2

Embryo Splitting

History

Answer 2

Cloning is a natural phenomena of nature: identical twins, plants self pollination, earthworms cut in half

Has Dreisch cloned sea urchins in late 1800s

Hans Spemman used a human hair to seperate a 2 celled salamander embryo

1952 - frog embros were split - some develop to tadpole stage but not developed to adutls

1983 - mouse embryos split - created viable embryos but no pups

Equine and bovine embryos were being split by the late 1980s

Tetra - a rhesus monkey was the first primate clone

Question 3

Embryo Splitting

Procedure

Answer 3

Egg and sperm are joined to form a fertilized egg

When embryo reaches about 8-cell stage it is split one/more times

At this stage, the embryo is a blastocyst and has not hatched from zona pellucida

Only very good quality embryos are split

A micromanipulator scope, vacuum tips, and microblades are used

Pieces of the original embryo are placed in empty ZP

Embryos transferred into a recipent that is on the same day post ovulation as the donor

The survivability of split embryos is less than un-manipulated embryos - but not too bad

Question 4

Cloning (Nuclear Transfer)

Answer 4

Bovine, ovine, canine, and equine

Question 5

Nuclear Transfer

Answer 5

Steen Willadsen - 1986: cloned lambs using cells from sheep embryos

Allowed for multiple cloned embryos

NT is the transfer of the nucleus of a donor embryonic cell to another cell

Recipient cells are oocytes whose chromosomes have been removed

OOcytes arrested in metaphase 2

Removal of maternal DNA is done with microtools

Donor cells must often come from embyonic cells form the blastocyst stage

Question 6

Granada Genetics

Answer 6

First cattle cloning company

Production of identical bovine offspring by nuclear transfer using embryonic cells

Bu what’s the limitation of cloning from embryos

Only cloning the potential of a single embryo, but you don’t really know how valuable the adult animal is going to be

Question 7

Cloning

Somatic Cell Nuclear Transfer

Answer 7

Ovine, Canine, bovine, and equine

Multiple genetically identical animals can be produced by taking an adult cell and “re-programming” it to allow differentiation into an entire organism when implanting into enucleated eggs

Question 8

Somatic Cell Nuclear Transfer

Donor Cell

Answer 8

Biopsy from ear of animal to be cloned

Question 9

Somatic Cell Nuclear Transfer

Recipient Cells

Answer 9

Eggs collected from slaughterhouse ovaries

Question 10

Cloned Calf Syndrome

Answer 10

We don’t fully understand why these embryos / fetuses / calves are not the same as natural pregnancies

Natural Pregnancy: 1-3% pregnancies lost in 1st trimester

IVF pregnancies: 5% loss in cattle, 11% loss in sheep

SCNT Clone pregnancies: 70% pregnancies lost in first trimester - up to 95% total loss

Culture conditions may be involved

Placenta may not develop normally - inappropriate transition from yolk sac to allantoic nutrtion

Failure of normal placentome development

Question 11

Cloned Calf Syndrome

Calves that complete gestation

Answer 11

Frequently increased birthweight

Abnormal placenta - placental edema

Few/Huge placentomes

Normally, fetal growth is contrained by maternal and placental factors

Increased placental origin IGF-1

Repirartory Distress Syndrome

pulmonary hypertension

Pood adrenal gland development / function

Low fetal cortisol lead to insufficient lung surfactant

Treatment of recipient dam with cortisol prior to delivery and oxygen therapy in the neonatal period