1. CLONING: NEW FRONTIERS OF ANIMAL REPRODUCTIVE BIOTECHNOLOGY Dr. Gatot Ciptadi Lab. Of Genetics and Animal Breeding Fac of Animal Sci., UB LSIH-UB

2. BIOTEKNOLOGI REPRODUKSI MAMPU MENYELAMATKAN SUMBER GENETIK HEWAN LANGKA GT Introduction somatis oosit sperma

3. Basic technique for nuclear transfer (cloning) 1. Cryopreservation (Freezing) Seeding, Stepwise, Direct, Vitrification 2. Embryo transfer (ET) Superovulation Collection of embryos (Flushing) Evaluation of embryos Synchronization of estrus 3. In vitro fertilization (IVF) Collection of oocytes from ovaries In vitro maturation In vitro fertilization In vitro culture 4. Micromanipulation Sperminjection (ICSI; IVM, Activation, IVC) Clone (Nuclear transfer) IVM, IVF, IVC Enucleation, Nuclear injection Fusion, Activation Gene transfer ( vector, Electropolation) Gene transfer (Injection into zygotes) Basic technique for nuclear transfer (cloning) 1. Cryopreservation (Freezing) Seeding, Stepwise, Direct, Vitrification 2. Embryo transfer (ET) Superovulation Collection of embryos (Flushing) Evaluation of embryos Synchronization of estrus 3. In vitro fertilization (IVF) Collection of oocytes from ovaries In vitro maturation In vitro fertilization In vitro culture 4. Micromanipulation Sperminjection (ICSI; IVM, Activation, IVC) Clone (Nuclear transfer) IVM, IVF, IVC Enucleation, Nuclear injection Fusion, Activation Gene transfer ( vector, Electropolation) Gene transfer (Injection into zygotes)

4. Cloning Techniques Cloning Techniques Desection of morula and blastocyst (Embryo) Desection of morula and blastocyst (Embryo) Deaggregation (Isolation) of blastomeres (Embryo) Deaggregation (Isolation) of blastomeres (Embryo) Clone Clone Micromanipulation Micromanipulation Nuclear transfer Nuclear transfer Micromanipulation +Gene transfer Micromanipulation +Gene transfer 1. Desection of morula and blastocyst (Embryo) 1. Desection of morula and blastocyst (Embryo) 2. Deaggregation (Isolation) of blastomeres (Embryo) 2. Deaggregation (Isolation) of blastomeres (Embryo)

5. DREAMS SOMETIMES COME TRUE ! Bioteghnology has the possibility to be revived a scare and an extinct animal (Film Jurasic Park) The birth of the clone animals is great influencing the frontier of research of animal reproductive biotechnology Why ? Somatic cell (deferentiated) was reprogramed back to embryonal stage and resulted in normal offsprings.

6. GT Bioteknologi Reproduksi: Sumbangan KLONING Pada Ilmu Pengetahuan GT

7. Nuclear transfer Activation ET Invitro maturation Surrogate mother pig S u m m a r y Recipient oocytes Donor cell Culture of Cloned embryos In vitro culture Enucleation

8. Introduction of donor cell Enucleation Passages of somatic cells Activation Enbryo transfer Production of transgenic piglets In vitro Maturation of oocytes Introduction of Extraneous gene Xenotransplatation Production of human protein human protein Synchronization of estrus Surrogate mother of miniature pigs Nuclear trasfer Recipient oocytes Donor cell derived from miniature pigs Selection of M 2

9. Nuclear transfer procedure to produce reconstructed embryo: inject donor cell in the perivittelin space of enuclated oocyte the performed fusion cell is performed intracytoplasmic direct nuclear injection (IDNI) activated artificially

10. Enucleated Non enucleated IDNI Enucleation TECHNICAL ASPECT: TN

11. 1 2 3 4 A B Sel Hidup… / Mati… 1. Besar, 2. Sedang, 3. Kecil: 22.16 % 4.Abnormal S e l e k s i S e l D o n o r Tanpa Aktivasi Aktivasi tunggal Ukuran sel

12. DEVELOPMENT OF NEW TECH. IN REPROD ANIMAL 11997Birth of dolly (1 st animal cloning using somatic cells) 21998Birth of cows: Charlie/George: ( serum albumin) Specific protein for human blood aglutination 32000Pig cloning (transgenic) for organ transplantation (human), 4.2001Inter species nuclear trasnfer (Yak to Bos Taurus) advantages: non conventional product of livestock : -Genetic quality improvement -Biopharmacies -Organ Transplantation (Biomedic industry) -Genetic concervation of endangered animals

13. Genetic Consideration and Constraints of Gen Manipulation???: No.PersilanganFertilitasKeunggulan F1 JantanBetina 1.BantengX Zebu-+Laju pertumbuhan 2.Keledai Xkuda-+Kerja + ketahanan 3.Sapi x Yak--BB/Power/Susu 4.Bos taurusxB. Indicus++Daging 5.Entog X Itik??Daging /pertb. Catatan: Bukan penghasil bibit Secara teknis memungkinkan, tetapi Tujuan utama bukan genetik.

14. Have a nice dream……… Dr. Tanaka H.