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

Generation of transgenic non- human primates with

germline transmission

Creation of 'GM' monkey heralds health revolutionGene breakthrough offers hope of treatments for 'incurable' Parkinson's disease and MSBy Steve Connor, Science EditorThursday, 28 May 2009

Glowing Green Monkeys Illustrate Important but Controversial AdvanceBy Rob SteinWashington Post Staff Writer Thursday, May 28, 2009

Posted: Thu, May 28 2009. 12:19 AM ISTAre monkeys the new lab rats for genetic tests?Jacob P. Koshy

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 ‘viralvector’; 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 ResDOI 10.1007/s11248-009-9316-6Review

INTRODUCTIONWhy 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.

COMMON MARMOSET

Callithrix jacchus New world primate Small size Sexual maturity at 12-18

months Short Gestation

period(144 days) Females have 40-80

offspring during their life

STRATEGY1) 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

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.

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.

RESULT AND OBSERVATIONS

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).

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

2) EGFP transgene integration in the genomeExamined 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.

584 587 588 594 666 584 588 594/666 584 587 588 594 666

Blood

Placenta Fibroblast

23,1309,416

6,557

4,361

23,1309,4166,557 4,361

23,130

9,4166,557

Figure 2 ) Transgene insertions in several infant tissues. Southern blotanalysis.

4,361

Figure 3. Karyograms for 584 animals from FISH analysis

Figure 4. Karyograms for 587 animals from FISH analysis

Figure 5. Karyograms for 588 animals from FISH analysis

Figure 6. Karyograms for 594 animals from FISH analysis

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

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

3)Expression of the EGFP transgeneEGFP 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

584 587 588 594 666 -ve

EGFP

EGFP

EGFP

β-actin

β-actin

β-actin

584 588 594/666 -ve

Figure 9. RT–PCR results from different tissues

Hair root

Blood

Placenta

Figure 10. Immunohistochemical and epifluorescent analyses of frozen placenta.

584 584

588 588

594/666

594/666

-ve control -ve control

Figure 11. Immunohistochemical and epifluorescent analyses of frozen ear tissue.

584

587587

594594

666 666

584

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.

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.

Sperm Embryo666 666 584 wtEGFP

EGFP RT-

β-actin

Placenta Hair

wt 687 wt 687

687687 wtwtRT+ RT -

Skin

EGFP

β-actin

β-actin

EGFP

687 wt

a)

b)

c)

d)

e)

IVF CAG CMV FE-1 TOTAL

Total Lentiviral injectionsGFP expression confirmedGFP expressionGFP expression Rate (%)Embryo transferNumber of surrogatesNumber of pregnanciesRate of pregnancy(%)Number of deliveriesBirthsNumber of TgProduction Rate(Tg/inj)

Suc+ Suc- 11 7 11 5 8 4 72.7 80 8 5 3 5 1 0 33.3 0 1 0 1 0 1 0 9.0 0

Suc+ Suc-5 05

06

0 60.0

0.07

0 1

00

00

00

00

00

00

0

Suc+ Suc-0 4 0 2 0 2 0 50.0 0 4 0 4 0 0 0 0 0 0 0 0 0 0 0 0

Suc+ Suc-16

11 16

7 11

6 68.8

85 10

9 4

9 1

0 25.0

0 1

0 1

0 1

0 6.3

0

Total272317 73.9 19 13 1 7.7 1 1 1 3.7

NATURALTotal Lentiviral injectionsGFP expression confirmedGFP expressionGFP expression Rate (%)Embryo transferNumber of surrogatesNumber of pregnanciesRate of pregnancy(%)Number of deliveriesBirthsNumber of TgProduction Rate(Tg/inj)

28 2 24 2

23 1 82.1

50.0 26 1 14 1 4 0 28.6

0.0 2 0 2 0 2 0 7.1

0.0

4 2

4 24 2100.0

100.05 2 2 1 1 0 50.0

0 1 0 2 0 2 0 50.0

0

17 11 16 416 4100.0 100.0 17 1112 7 1 0 10.7 0 0 0 0 0 0 0 0 0

49 15 44 8 43 7 97.7 87.5 47 14 28 9 6 0 21.4 0 3 0 4 0 4 0 8.5 0

64 52 50 96.2 61 37 6 16.2 3 4 4 6.3

Efficiency of lentiviral injection

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

FUTURE PERSPECTIVESTo 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.

“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.

Scientists involved

Sasaki Erika

THANK YOU

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