Generation of transgenic non- human primates with germline transmission

1. Generation of transgenic non- human primates with germline transmission Erika Sasaki, Hiroshi Suemizu, Akiko Shimada, KisaburoHanazawa, Ryo Oiwa1, Michiko Kamioka, IkuoTomioka, Yusuke Sotomaru, Reiko Hirakawa, TomooEto, Seiji Shiozawa, Takuji Maeda, Mamoru Ito, Ryoji Ito, Chika Kito, Chie Yagihashi, Kenji Kawai, Hiroyuki Miyoshi, YoshikuniTanioka, Norikazu Tamaoki, SonokoHabu, Hideyuki Okano4 & Tatsuji NomuraNature 28th May 2009

2. Creation of 'GM' monkey heralds health revolution Gene breakthrough offers hope of treatments for 'incurable' Parkinson's disease and MS By Steve Connor, Science EditorThursday, 28 May 2009 Glowing Green Monkeys Illustrate Important but Controversial Advance By Rob Stein Washington Post Staff Writer Thursday, May 28, 2009 Posted: Thu, May 28 2009. 12:19 AM IST Are monkeys the new lab rats for genetic tests? Jacob P. Koshy

3. Transgenic Res DOI 10.1007/s11248-009-9316-6 Review ‘‘What’s wrong with my monkey?’’ Ethical perspectives on germline transgenesis in marmosets I. Anna S. Olsson Æ Peter Sandøe The reasons for the enthusiasm over the monkey born this year are three: it carries a transgene inherited from its genetically modified parents; the parents were modified by means of a so-called ‘viral vector’; and the monkey was a marmoset.

4. INTRODUCTION • Why non-human primates as models? • Thousands of non-human primates are used around the world in research. • Striking similarities between nonhuman primate species and human beings. • The development of drugs and vaccines.

5. COMMON MARMOSET • Callithrixjacchus • New world primate • Small size • Sexual maturity at 12-18 months • Short Gestation period(144 days) • Females have 40-80 offspring during their life

6. STRATEGY 1) Vector • Self-inactivating HIV-1 type • Tagged with CMV-EGFP , CAG-EGFP and EF1-α-EGFP 2) IVF embryos • Recombinant human follicle stimulating hormone and human chorionic gonadotropinwas administered by intramuscular injection . • They anaesthetized and follicular aspiration was performed surgically. • Oocytes were treated Hyaluronidase and incubated . • Ejaculated semen was collected. • IVF was performed. • lentiviralvector injection was performed by EppendorfFemtoJet express and a Narishigemicromanipulator . • Injection at pronuclear to morula stage.

7. 2) Natural embryos • Natural embryo collection. • Embryos were treated with 0.25 sucrose. • lentiviralvector injection using an EppendorfFemtoJet express and a Narishige micromanipulator. • The embryos were cultured until GFP expression was confirmed. • EGFP-expressing embryos were transferred to recipients. • Pregnancy test of the recipients by plasma progesterone analysis.

8. RESULT AND OBSERVATIONS

9. 1)Production of transgenic marmosets • Seven recipients were pregnant. • Three recipients miscarried and the other four delivered five healthy offspring (three singletons & one pair of twins). • One male (number 666) and four females, on days 144–147 after ovulation were born. • Named as Hisui(584), wakaba(587), Bankao(588), and twin infants Kei(594) / Kou(666). • The EGFP transgene was driven by the CAG promoter in three newborns (584, 587 and 588) and by the CMV promoter in the other two (594 and 666).

10. The transgenic marmoset infants a b c d Figure 1) Shown are 584 (Hisui) (a), 587 (Wakaba) (b), 588 (Banko) (c), and twin infants 594(Kei)/666 (Kou) (d) All animals except 588 expressed EGFP in their paw. 666 expressed EGFP at a slightly lower level

11. 2) EGFP transgeneintegration in the genome • Examined placenta, hair roots, skin and peripheral blood cells. • Only three placentae584, 588 and that shared by twins 594/666) could be collected. • Infant 588 showed transgene integration only in the placenta.

12. 584 587 588 594 666 584 588 594/666 584 587 588 594 666 23,130 23,130 23,130 9,416 9,416 9,416 6,557 6,557 6,557 4,361 4,361 4,361 Blood Placenta Fibroblast Figure 2 ) Transgene insertions in several infant tissues. Southern blot analysis.

13. Figure 3. Karyograms for 584 animals from FISH analysis Figure 4. Karyograms for 587 animals from FISH analysis

14. Figure 5. Karyograms for 588 animals from FISH analysis Figure 6. Karyograms for 594 animals from FISH analysis

15. Figure 7. Karyogram for 666 male type MNCs from FISH analysis Figure 8. Karyogram for 666 female type MNCs from FISH analysis

16. 3)Expression of the EGFP transgene • EGFP messenger RNA was detected in the hair roots of all the infants except 588 ,in the peripheral blood cells of 584 and 587, by RT–PCR & in all of the placental samples, 584, 588 and 594/666

17. 584 587 588 594 666 -ve EGFP Hair root β-actin EGFP Blood β-actin 584 588 594/666 -ve EGFP Placenta β-actin Figure 9. RT–PCR results from different tissues

18. 584 584 588 588 594/666 594/666 -ve control -ve control Figure 10. Immunohistochemical and epifluorescent analyses of frozen placenta.

19. 584 584 587 587 594 594 666 666 Figure 11. Immunohistochemical and epifluorescent analyses of frozen ear tissue.

20. 4) Germline transmission of the transgene • Two of the animals (666 and 584) became sexually mature. • The transgene expression in their gametes was analyzed. • RT–PCR analysis demonstrated the presence and expression of the transgene in the germ cells of 666. • The IVF embryos produced using semen collected from 666 and wild-type oocytes strongly expressed EGFP by fluorescence microscopy. • one of three pre-implantation embryos collected from 584 strongly expressed EGFP.

21. Three EGFP-positive IVF embryos from the male animal (666) were then transferred into a surrogate mother. • One neonate (687) was delivered at full term by caesarean section. • The neonate expressed the transgene.

22. Sperm Embryo c) a) 666 666 584 wt EGFP EGFP RT- β-actin b) Placenta Hair 687 wt 687 wt EGFP β-actin d) Skin RT+ RT - 687 wt 687 wt EGFP β-actin wt 687 e)

23. Efficiency of lentiviral injection

24. Discussion • The first report of transgenic non-human primates showing germline transmission of the transgene. • It’s effective for increasing the birth rate and reducing the number of surrogate mother. • Advantageous to use marmoset natural embryos. • Production rate was high in sucrose treated embryos. • Technique is sufficiently effective for the production and use of genetically modified marmosets as human disease models

25. FUTURE PERSPECTIVES • To enable the introduction of larger transgenes into marmoset embryos. • Targeted gene-knockdown marmosets development using RNA interference (RNAi) lentiviruses to study human diseases involving the malfunctions of specific genes. • To obtain genetically modified non-human primate models for translational research, investigations of regenerative medicine and gene therapy, and clarification of the scientific gaps among transgenic mice, human disease models, and real human diseases.

26. “Take your stinking paws off me, you damned dirty ape!”  CharletonHeston's memorable line from the 1968 classic movie Planet of the Apes is about a world where chimpanzees, orangutans, and gorillas – genetically modified in the 2001 version of movie – enslave humans. The original version was a story built around the racial intolerance, oppression, and animal experimentation of the late 1960s within a story of alternative evolution.

27. Sasaki Erika Scientists involved

28. THANK YOU THANK YOU