1. Generation of transgenic non- human primates with germline transmission
Erika Sasaki, Hiroshi Suemizu, Akiko Shimada, Kisaburo Hanazawa, Ryo Oiwa1, Michiko Kamioka, Ikuo Tomioka, Yusuke Sotomaru, Reiko Hirakawa, Tomoo Eto, Seiji Shiozawa, Takuji Maeda, Mamoru Ito, Ryoji Ito, Chika Kito, Chie Yagihashi, Kenji Kawai, Hiroyuki Miyoshi, Yoshikuni Tanioka, Norikazu Tamaoki, Sonoko Habu, Hideyuki Okano4 & Tatsuji Nomura Nature 28th May 2009

2. Are monkeys the new lab rats for genetic tests?
Creation of 'GM' monkey heralds health revolution
Gene breakthrough offers hope of treatments for 'incurable' Parkinson's disease and MS
By Steve Connor, Science Editor Thursday, 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 :19 AM IST
Are monkeys the new lab rats for genetic tests?
Jacob P. Koshy

3. 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.
‘‘What’s wrong with my monkey?’’ Ethical perspectives on germline transgenesis in marmosets
I. Anna S. Olsson Æ Peter Sandøe
Transgenic Res
DOI /s
Review

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 Callithrix jacchus New world primate Small size
Sexual maturity at months
Short Gestation period(144 days)
Females have offspring during their life

6. STRATEGY 1) Vector 2) IVF embryos 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 gonadotropin was 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.
lentiviral vector injection was performed by Eppendorf FemtoJet express and a Narishige micromanipulator .
Injection at pronuclear to morula stage.

7. 2) Natural embryos Natural embryo collection.
Embryos were treated with 0.25 sucrose.
lentiviral vector injection using an Eppendorf FemtoJet 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).
Sher bhadur paudel

10. The transgenic marmoset infantsb 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 transgene integration in the genome
Examined placenta, hair roots, skin and peripheral blood cells.
Only three placentae 584, 588 and that shared by twins 594/666) could be collected.
Infant 588 showed transgene integration only in the placenta.

12. Blood Placenta Fibroblast/
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

14. Figure 5. Karyograms for 588 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. ve
EGFP
β-actin
/ ve
Figure 9. RT–PCR results from different tissues
Hair root
Blood
Placenta

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

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

23. Efficiency of lentiviral injection IVF CMV FE-1
CAG
CMV
FE-1
TOTAL
Total Lentiviral injections
GFP expression confirmed
GFP expression
GFP expression Rate (%)
Embryo transfer
Number of surrogates
Number of pregnancies
Rate of pregnancy(%)
Number of deliveries
Births
Number of Tg
Production Rate(Tg/inj)
Suc Suc-
Suc+ Suc-
Suc Suc- 11
Total 27 23 17
73.9 19 13 1
7.7
3.7
NATURAL 2 64 52 50
96.2 61 37 6
16.2 3 4
6.3

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
“Take your stinking paws off me, you damned dirty ape!”  Charleton Heston'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. Scientists involved
Sasaki Erika

28. THANK YOU
THANK YOU
THANK YOU