Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body

Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson’s disease and Alzheimer’s disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research.


Result
Production of GFP transgenic cynomolgus monkeys. In rodents, the pronuclear injection of DNA into fertilized eggs is an efficient strategy to generate transgenic animals. An alternative method is the use of genetically engineered embryonic stem cells. However, these strategies have been unsuccessful in NHPs to date. Since a transgenic marmoset with the capability of germ line transmission has been generated by injecting lentivirus into the perivitelline space of a two-cell-stage embryo 38 , we followed the technique to create a transgenic cynomolgus monkey.
Firstly, we constructed a lentiviral vector that carries GFP cDNA under the control of human cytomegalovirus early enhancer and chicken beta actin (CAG) promoter (Fig. 1A), because CAG promoter is able to achieve ubiquitous expressions in various species 50 . Transfer of five lentivirus-injected embryos into three recipients resulted in two pregnancies, one of which ended in a miscarriage of twins on day 92 of gestation (Table 1). Detailed organ examination and histology identified no abnormalities (data not shown). Although one of the twins (#1) showed no detectable fluorescence, the other (#2) showed strong fluorescence in the face, skin, placenta and brain (Fig. 1B, data not shown). Strong fluorescence was observed in the organs of #2 foetus including the umbilical cord, placenta and amnion compared with #1 foetus under a fluorescent stereomicroscope (Fig. 1C). PCR analysis was performed to detect GFP transgene and demonstrated that the GFP transgene was integrated ubiquitously in the genome of all tissues in #2 foetus, but not #1 foetus. Consistent with this, RT-PCR analysis indicated that GFP mRNA was expressed ubiquitously in #2 foetus, but not #1 foetus (Fig. 1D). From these results, the #1 and #2 foetuses were determined to be wild type (WT) and transgenic (Tg), respectively. Abundant GFP mRNA expression was detected in the brain, heart and pancreas in #2 foetus, while moderate GFP expression was detected in the kidney, spleen and amnion by RT-quantitative PCR (RT-qPCR) (Fig. 1E). We confirmed GFP protein expression in these organs by immunohistochemistry with an anti-GFP antibody and found that fluorescence was detected in all these organs from the #2 foetus ( Fig. 2A). Strong fluorescence was observed in the heart, lung, spleen and stomach from #2 foetus, but not #1 foetus or the miscarried WT foetus ( Fig. 2A, Supplementary Fig. 1A), under the same instrumental settings (same laser intensity, please see Materials and Methods). However, we detected moderate fluorescence in the liver and kidney in #1 foetus that was WT as indicated by PCR analysis (Fig. 2A). To investigate carefully the nature of the fluorescence, we examined tissues from the liver and kidney of another miscarried WT foetus and found that these organs also had comparable fluorescence ( Fig. 2A). Careful comparison revealed significant differences in the intensity and pattern of fluorescence between WT and #2 foetus, demonstrating that the fluorescence observed in the tissues of #1 and WT foetuses were autofluorescence and that the fluorescence in the #2 foetus was derived from GFP protein. Regarding autofluorescence, as laser power increased, autofluorescence of hepatocytes in the liver and renal tubules in the kidney also increased ( Supplementary Fig. 1B). WT monkey face exhibited moderate autofluorescence as if it is GFP transgenic monkey ( Supplementary Fig. 1C), showing the importance of negative control. Taken together, we concluded that it was very important to compare fluorescence of WT and Tg at the same time, and #2 foetus was Tg with ubiquitous overexpression of the GFP protein.   GFP expression in the brain tissue. To investigate the detailed expression of GFP in the brain of a day 92 foetus, we examined the colocalisation of GFP, a radial glial and astroglial marker, glial fibrillary acidic protein (GFAP), and a neuronal specific nuclear protein marker, NeuN (Fig. 2B). Widespread GFP expression was observed throughout the cortex, including the forebrain, midbrain, and hindbrain. At a higher magnification, neuronal GFP expression was identified by its colocalisation with NeuN in the prefrontal cortex (Fig. 2B). GFP and GFAP were also colocalised in the lateral ventricle (Fig. 2B).

Germline transmission of the transgene.
To investigate whether the transgene was transmitted to the germline, we examined the colocalisation of GFP and VASA (DDX4), a marker for primordial germ cell (PGC) of the ovary in the day 92 foetus. GFP and VASA were clearly colocalised in a subset of the ovary cells (Fig. 2C). Expression of OCT4, another marker for PGC was also well overlapped with that of GFP (Fig. 2C).
Partial GFP expression in the peripheral blood and fibroblasts. Previously, GFP transgenic marmoset showed partial GFP expression ranging from 0 to 19.1% in the whole peripheral blood 38 . To estimate the percentage of GFP positive cells in the peripheral blood of #2 foetus, immunohistochemistry with an anti-CD45 antibody, a marker for white blood cells, was performed on the spleen and showed that 55% of CD45 positive cells expressed GFP protein (Fig. 3A), indicating that peripheral blood cells only partially expressed GFP.
To characterize GFP positive and negative cells, we established fibroblasts from the #2 foetus tail and found that about 60% of the cells were GFP-positive by FACS analysis (Fig. 3B,C). Interestingly, we found that GFP-negative cells also carried GFP transgene, suggesting that the transgene was not transcribed (Fig. 3C). Previous studies indicated that lentiviral transgene often undergo silencing through CpG methylation, and that treatment with valproic acid, a histone deacetylase inhibitor, was effective to rescue silenced gene transcription 51 . When GFP-negative fibroblasts were cultured in the presence of valproic acid, a significant number of GFP-positive cells were observed, indicating that GFP silencing may be caused by histone deacetylation (Fig. 3D). Taken together, a GFP cynomolgus monkey foetus was successfully obtained, yet it only showed partial GFP expression and silencing of the transgene.

Optimization of lentivirus infection on GFP expression.
To generate a transgenic cynomolgus monkey that expressed GFP homogenously in each of the various tissues, we optimized the lentivirus injection protocol. Since the lentivirus solution had a carryover of GFP protein contaminated in the process of virus production ( Fig. 4A), we purified the lentivirus solution further by centrifugation in a sucrose cushion to remove GFP protein and obtained the highly purified lentiviral solution (Fig. 4A).
Wongsrikeao et al., reported that when virus was injected into mature oocytes before fertilization, uniform infection was achieved in a cat embryo 46 . Therefore, matured oocytes were subjected to perivitelline-space injection with lentivirus 4 h before intracytoplasmic sperm injection (ICSI) (PreI) (Fig. 4B). After the injection, the oocytes were cultured for 4 h and subjected to ICSI and cultured until blastocyst stage (day 8).

Full-term development of GFP cynomolgus monkeys. An offspring (named PreI Tg #1) from the
PreI embryo was born successfully and showed strong fluorescence in the face and placenta compared with that of WT offspring (Fig. 4C,D). Amnion of PreI Tg #1 monkey showed homogenous GFP expression at the cellular level (Fig. 4E). PCR analysis was performed to detect GFP transgene and demonstrated that the GFP transgene was integrated in the genomic DNA from umbilical cord and placenta (Fig. 4F), demonstrating that PreI Tg #1 monkey is a GFP transgenic.
Although PreI Tg #1 monkey clearly showed strong GFP fluorescence in the skin and homogenous GFP expression in the amnion at the cellular level, GFP expressions in the tissues were not addressed due to consideration for animal welfare. Another offspring (named PreI Tg #2) was born, but died 3 days after birth ( Table 2). Hypoplasia of the pituitary grand and pancreas were observed at the necropsy (data not shown). The whole body of the transgenic cynomolgus monkey showed strong fluorescence under an excitation light (Fig. 5A). The fluorescence was also detected in the placenta (Fig. 5A). PCR analysis indicated that GFP lentiviral transgene was integrated into the genome of various tissues (Fig. 5B). RT-PCR analysis indicated that GFP was expressed abundantly at the mRNA level in the brain, lung, kidney and stomach, while moderate GFP expression was observed in the heart, liver, pancreas, spleen, intestine and testis (Fig. 5B,C). Immunohistochemistry revealed abundant GFP expression in all tissues tested including the heart, lung, liver, kidney, spleen, stomach, and intestine (Fig. 6A, Supplementary Fig. 2A,B). This was in sharp contrast with the autofluorescence observed in WT tissues (Fig. 6A). GFP protein expressions were also evident in lateral ventricle and grey matter layer (Fig. 6B). To investigate the contribution of the transgene to the germ cell lineage, we examined GFP expression in PGCs and found that almost all VASA-positive PGCs expressed GFP (Fig. 6C). To examine the ratio of GFP positive cells in offspring, high magnification images of sections were analysed and showed that > 95% of cells were GFP positive (data not shown). Furthermore, CD45 positive peripheral blood was analysed and 99% of CD45 positive cells expressed GFP (Fig. 6D). To demonstrate this more clearly, fibroblasts were established from the tail and ear of offspring, and almost all cells were GFP-positive under microscopic observation (Fig. 6E). Consistent with this, FACS analysis of fibroblasts showed that nearly 100% of cells were GFP-positive (Fig. 6E). Collectively, these results demonstrated that the GFP cynomolgus monkey created by PreI technique expressed GFP homogenously in each of the various tissues.
Comparison of expression levels of GFP protein in tissues from Tg monkeys and the GFP mouse. The green mice that ubiquitously express GFP have been created and been used for many biomedical researches such as transplantation studies 52 . Although the generation of GFP rhesus monkey and GFP marmoset have been reported [36][37][38]49 previously, there was no study about the comparison of the level of GFP expression with that of the GFP mice. To evaluate the usefulness of GFP cynomolgus monkey established in this study, we compared GFP expression levels in tissues from GFP cynomolgus monkey with those from GFP mice 42 by western blot analysis. Strong GFP expressions were detected in lung, kidney and spleen of PreI Tg cynomolgus   monkey compared to those of GFP mouse (Fig. 7). #2 (Tg) foetus also expressed moderate level of GFP protein (Fig. 7). Thus, GFP cynomolgus monkeys generated in this study expressed even higher level of GFP protein in various tissues compared with those of GFP mice, and the technique established in this study will be very useful for the creation of human disease models.

Discussion
Transgenic NHPs will be valuable for human disease models, such as Parkinson's disease and Alzheimer diseases. So far, transgenic NHP models of Huntington's disease 37 and Parkinson's disease 53 have been created and reported. GFP is frequently used in biomedical research and GFP-expressing animals are an important source of transplantation studies. Previously, generation of GFP rhesus monkeys and GFP marmosets have been reported [36][37][38]49 , yet no GFP cynomolgus monkeys. To the best of our knowledge, this is the first study reporting the generation of GFP expressing cynomolgus monkeys. The strategy established in this study will be valuable for generating cynomolgus monkeys that homogenously overexpress a causative gene for human disease in each tissue. During the generation of GFP cynomolgus monkeys, we were aware that moderate fluorescence expression is detectable even in WT tissues. It was critically important to use WT tissues as a negative control to confirm the fluorescence derived from GFP protein in transgenic tissues. It is important to note that some previous reports of GFP transgenic monkeys showed only GFP transgenic tissues but not corresponding WT tissues 36,37,49 .
Our analysis indicated that when lentivirus was injected into a two-cell-stage embryo, partial GFP expression was achieved. Although lentivirus can transduce non-dividing cells, integration is significantly slowed by the nuclear membrane 48,54 . This has previously been observed in other species including marmosets 38 .This mosaicism may be caused by the delay of lentiviral integration into the genome. Interestingly, a bovine study showed that the lentiviral genome could not efficiently enter the pronucleus 48 . Wongsrikeao et al. examined the effect of infection timing and found mosaic expression was able to be avoided by injecting lentivirus into cat oocytes before IVF 46 . Consistent with this, when lentivirus was injected into cynomolgus monkey oocytes before fertilization, rather than the two-cell embryo stage, we obtained offspring with homogenous GFP expression in each tissue, showing that lentiviral injection into mature oocytes before fertilization is an effective way to create transgenic NHPs with homogenous expression of GFP in each of the various tissues, including the amnion, and fibroblasts.
NHPs are invaluable models for high order brain research into disorders such as Parkinson's disease, Autism and Alzheimer's disease. Alzheimer pathology is correlated strongly with the density of activated astrocytes 55 . In these cells, the expression of GFAP is strongly upregulated 56,57 . In Parkinson's disease patients, the phosphorylation level of GFAP at serine 13 was significantly lower compared with control subjects 58 . As the present study indicated that the GFP was expressed ubiquitously in various organs and cells including neuronal cells of transgenic cynomolgus monkeys, this transgenic technique will be useful to create human disease models for Parkinson's disease and Alzheimer's disease amongst others. The GFP cynomolgus monkey will also provide a source of green pre-implantation stage embryos, which can be used for the production of chimeric cynomolgus monkeys by the injection or aggregation with non-green embryonic stem cells.

Materials and Methods
Animals. Experimental procedures were approved by the Animal Care and Use Committee of Shiga University of Medical Science and methods were carried out in accordance with the approved guideline (Approval number: [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. Oocytes were collected from seven sexually mature female cynomolgus monkeys, aged 4-8 years and weighing 2.1-3.9 kg. Twenty sexually mature females aged 4 years old and weighing 2.0-3.8 kg, were used as recipients. Sperm were collected from one sexually mature male cynomolgus monkey, aged 12 years and weighing 6.2 kg. Temperature and humidity in the animal rooms were maintained at 25 ± 2 °C and 50 ± 5%, respectively. Monkeys were housed individually in cages (800 × 500 × 800 mm). The light cycle was 12 h of artificial light from 8 am. to 8 pm., Each animal was fed 20 g/kg of body weight of commercial pellet monkey chow (CMK-1; CLEA Japan, Inc., Tokyo, Japan) in the morning, supplemented with 20-50 g of sweet potato in the afternoon. Water was available ad libitum.
Oocyte collection. Ovarian stimulation and oocyte collection were carried out as previously described by Yamasaki et al. 59 with some modifications. In brief, beginning at menses, the level of sex steroid hormones was reduced by subcutaneous injection of 0.9 mg of a gonadotropin-releasing hormone antagonist (Leuplin; Takeda Chemical Industries, Ltd., Osaka, Japan). Two weeks later, Micro infusion pump (iPRECIO SMP-200, ALZET Osmotic Pumps Co., Cupertino, CA, USA) with 15 IU/kg human follicle-stimulating hormone (hFSH; Asuka Pharmaceutical Co., Tokyo, Japan) was embedded subcutaneously under anesthesia and injected 7 μ l/h for 10 days. On the day after the last hFSH injection, 400 IU/kg human chorionic gonadotropin (hCG; Puberogen, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) was injected intramuscularly. Oocytes were collected by follicular aspiration 40 h after hCG treatment, using a laparoscope (LA-6500, Machida Endoscope Co., Ltd., Chiba, Japan). Cumulus-oocyte complexes were recovered in Alpha modification of Eagle's medium (MP Biomedicals LLC, Solon, OH, USA), containing 10% Serum Substitute Supplement (SSS; Irvine Scientific, Santa Ana, CA, USA) at 38 °C, in an atmosphere of humidified 5% CO 2 and 95% in air for 1-2 h. Oocytes were stripped off cumulus cells by mechanical pipetting after brief exposure (< 1 min) to 0.5 mg/mL hyaluronidase (Sigma Chemical Co., St. Louis, MO, USA), adjusted with m-TALP (pH 7.4), a modified Tyrode solution, with, lactate, pyruvate, 0.3% bovine serum albumin (Sigma Chemical Co.) and HEPES. Then, oocytes were transferred to m-TALP without hyaluronidase, at 38 °C in 5% CO 2 until further use. Oocytes were classified by stages as germinal vesicle, metaphase I, metaphase II or degenerate. Western blot was performed with 2.5 μ g of proteins extracted from tissues of Tg monkeys, and the GFP mouse that carries one copy of the CAG promoter-GFP expression unit. Anti-GFP and β -actin antibodies were used to detect GFP and β -actin, respectively. Full-length blots are presented in Supplementary Fig. 3.
Scientific RepoRts | 6:24868 | DOI: 10.1038/srep24868 Intracytoplasmic sperm injection. Intracytoplasmic sperm injection was carried out on metaphase II-stage oocytes, as previously described 59 , in m-TALP containing HEPES (mTALP-HEPES). A glass needle (Humagen Fertility Diagnostics, Charlottesville, VA, USA), connected to an injector and an inverted microscope (Olympus Tokyo, Tokyo, Japan) with a micromanipulator, was used for sperm injection. Intracytoplasmic sperm injection was performed with fresh sperm collected by electric stimulation of the penis with no anaesthesia. Following ICSI, embryos were cultured in GIBCO CMRL Medium-1066 (Invitrogen, Carlsbad, CA, USA) supplemented with 20% bovine serum (Invitrogen) at 38 °C in 5% CO 2 and 5% O 2 then blastocyst stage embryos were generated in vitro. Embryo transfer and pregnancy detection. When embryos developed to expanded blastocysts, one or two embryos were transferred into each female recipient. Embryo transfer was performed as previously described 59 , with some modifications. Laparoscopy was carried out on alternate days from days 10-14 of the menstrual cycle after menstruation (ovulation day = day 0) until ovulation occurred. Recipients were selected for embryo transfer 1-6 day after ovulation. Neither the stage of the embryos nor the number of days after the recipient's ovulation were matched. Embryos were aspirated into a catheter (ETC3040SM5-17; Kitazato Medical Service Co., Ltd., Tokyo, Japan) under a stereomicroscope. The catheter was inserted into the oviduct of the recipient via the fimbria under the laparoscope, and the cultured embryo was transplanted with a small amount of medium. Pregnancy was determined by ultrasonography 30 days after ICSI.

FACS analysis.
Tail fibroblasts were established from day 92 foetuses (#1 and #2) and day 3 offspring (PreI Tg #2). After removal of tail skin, the remaining tails were washed in PBS and maintained in Dulbecco's modified eagle medium (DMEM, Invitrogen) containing 10% foetal bovine serum (FBS, JRH Biosciences, Corston Bath, UK), penicillin, streptomycin (Invitrogen), and primocin (Invivogen). Ear fibroblasts were established from PreI Tg #2 by cutting ears into 5 mm 3 fragments, washing in PBS and then cultured in DMEM containing 10% FBS, penicillin, streptomycin, and primocin. The fibroblasts were cultured until confluent and then collected by trypsin treatment. Cells were resuspended in a final volume of 500 μ l of PBS/2% FCS and analysed with a Becton Dickinson FACSAria cell sorter.
Observation of green fluorescence in transgenic offspring. Images were captured under 470 nm excitation light with 520-nm wavelength filters using a Nikon D3300 digital camera. No image intensifying procedures were applied to any of the images. A 1-day-old and 3-month-old cynomolgus monkeys were used as a negative control for PreI Tg #1 and #2 respectively. Western blot analysis. One mm 2 of tissues were incubated in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40 and 0.1% SDS) with protease and phosphatase inhibitors for 10 min at Scientific RepoRts | 6:24868 | DOI: 10.1038/srep24868 polyacrylamide gel at 250 V for 80 min and then transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked using Blocking One, and then incubated with rabbit anti-GFP AlexaFluor 488 conjugate (1:2,000) or rabbit anti-β -actin horseradish peroxidase (HRP) conjugate (1:4,000) antibody overnight at 4 °C in Blocking One. After three washes in Tween20-PBS (T-PBS), the membrane incubated with rabbit anti-GFP was treated with HRP-labeled anti-rabbit immunoglobulin G (1:2,000; Invitrogen) in Blocking One for 1 h at room temperature. After one wash of 15 min and five washes of 5 min each with T-PBS, peroxidase activity was visualized using the Chemi-Lumi One Super (Nacalai Tesque) according to the manufacturer's instructions. Tissues from a GFP mouse (C57BL/6-Tg(CAG-EGFP)10sb/J) 42 that carries one copy of the CAG promoter-GFP expression unit were provided by Drs. H. Kojima and M. Katagi (Shiga University of Medical Science) and used as a positive control.

Statistical analysis.
Statistical analyses of all data comparisons were carried out using the t-test using Excel software. P < 0.05 was considered statistically significant.