Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Qiang Sun ( qsun@ion.ac.cn ).

Healthy female cynomolgus monkeys (Macaca fascicularis), ranging in age from 5 to 12 years, were selected for this study. All animals were housed in the sunny room. The use and care of animals complied with the guideline of the Animal Advisory Committee at the Shanghai Institute of Biological Science, Chinese Academy of Sciences. The ethics application entitled “Reproductive physiology of cynomolgus monkeys and establishment transgenic monkeys” (#ER-SIBS-221106P) was approved by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences.

Method Details

Preparation of Nuclear Donor Cells Primary fibroblasts were isolated from a 61 day-old freshly aborted cynomolgus fetus. The fetal tissue were cut into small pieces after removing the head, tail, limbs, and viscera and digested with DNase (1 mg/ml) and collagenase IV (0.5 mg/ml) in cell culture medium containing DMEM supplemented with 100 IU/ml penicillin and streptomycin, 10% FBS, non-essential amino acids, and glutamine at 37°C in 5% CO 2 for 4 h. Isolated monkey fibroblasts were cultured in 10 cm dishes for 10-20 h until they reached confluency, disaggregated by trypsin, and stored in liquid nitrogen in cell culture medium containing 10% dimethylsulfoxide. Frozen fibroblasts (P0-P5) were thawed and cultured until confluency, and used for SCNT 3-7 days later. For preparation of adult monkey cumulus cells, fresh cumulus cells were collected from the follicular aspirates of oocyte donors and washed twice with TH3 medium. Re-suspended cumulus cells were used directly for SCNT.

Superovulation and Oocyte Collection Healthy female cynomolgus monkeys with regular menstrual cycles were chosen for superovulation and laparoscopy was used for oocyte collection. From day 3 of menstrual cycle, 25 IU recombinant human follitropin was injected intramuscularly twice daily for 7–8 days. On day 11, 1000 IU of human chorionic gonadotrophin (hCG) was also injected, oocytes were aspirated from follicles 2–8 mm in diameter 36 h later. The collected oocytes were cultured in the pre-equilibrated hamster embryo culture medium 9 (HECM-9) medium. Metaphase II-arrested oocytes were selected for manipulation.

Monkey SCNT and ICSI Procedures For monkey SCNT, a group of 15-20 monkey MII oocytes were transferred to manipulation drops containing HEPES-buffered Tyrode’s lactate medium (TH3) with 5 μg/ml cytochalasin B in a glass bottom dish. The spindle-chromosome complex was removed rapidly by a piezo-driven pipette under a spindle imaging microscopic system (Oosight) within 10 min for the entire group of oocytes. The enucleated oocytes were transferred to pre-equilibrated HECM-9 at 37°C under 5% CO 2 . After all oocytes were enucleated, a group of 15-20 enucleated oocytes were re-transferred to manipulation drops with TH3 and 5 μg/ml cytochalasin B. Fibroblasts or cumulus cells were briefly incubated in a medium containing fusogenic viral envelop HVJ-E and introduced to the perivitelline space of the enucleated oocytes by a micropipette through a slit in the zona pellucida that was created by laser irradiation. At 1-2 h after cell fusion, reconstructed embryos were activated in TH3 medium with 5 μM ionomycin for 5 min and transferred to HECM-9 medium containing 2 mM 6-dimethylaminopurine for 5 h. TSA (10 nM) were applied for 10 h during and after activation. For Kdm4d mRNA injection, 10 pl of 1000 ng/μl Kdm4d mRNA were used for injection at 6 h after activation with a piezo-driven micromanipulator (Primetech). It is possible that the Kdm4d mRNA concentration we used was not optimal and further optimization of the concentration is needed. Liu et al., 2016b Liu Z.

Nie Y.H.

Zhang C.C.

Cai Y.J.

Wang Y.

Lu H.P.

Li Y.Z.

Cheng C.

Qiu Z.L.

Sun Q. Generation of macaques with sperm derived from juvenile monkey testicular xenografts. 2 . For monkey intracytoplasmic sperm injection (ICSI), previously published procedure was used (). Briefly, a single picked sperm was injected into the ooplasm using a piezo-driven micromanipulator and fertilization was confirmed 6 h later by the presence of two pronuclei. Embryos were cultured in pre-equilibrated HECM-9 at 37°C under 5% CO

Culture of Monkey Embryos and Embryos Transfer Monkey ICSI and SCNT embryos were cultured in HECM-9 medium at 37°C under 5% CO 2 . The embryos were transferred to HECM-9+5% FBS medium after reaching 8-cell stage and the medium was changed every other day until the embryo reached the blastocyst stage. For embryo transfer, females with synchronous menstrual cycle whose ovaries had a stigma or fresh corpus luteum were used as surrogates. Embryos (at 2-cell to blastocyst stage) were transferred to the oviduct.

Genetic Analysis of Cloned Monkeys For short tandem repeats (STR) analysis, ear tissue sample collected from monkeys were used to extract DNA. Locus-specific primers each containing a fluorescent dye (FAM/HEX/TMR) were used for PCR amplification in batches. FAM, HEX or TMR-labeled STR amplicons were diluted and mixed with internal size standard ROX500 and deionized formamide, followed by capillary electrophoresis on ABI PRISM 3730 genetic analyzer to obtain the raw data. Sequencer-generated raw data were analyzed with the program Gene Marker 2.2.0, which produces wave plots, Excel documents (including information such as size and genotype), and DNA profiles. For single nucleotide polymorphism (SNP) analysis, ear tissue sample collected from monkeys were used to extract DNA. PCR with specific primers (F: CCACTTCACATCAAACCATCACTT, R: CAAGCAGCGAATACCAGCAAAA) in mtDNA were performed. DNA was amplified with 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by a 5-min extension at 72°C. The PCR products were used for sequencing and the result were used for the SNP analysis.

Kdm4d In Vitro Transcription For Kdm4d in vitro transcription, CDS of human Kdm4d gene was cloned from the cDNA plasmid provided by Dr. J.H. Han of Xiamen University. The DNA template was amplified by the T7 promoter containing primer (F: TTAATACGACTCACTATAGGGATGGAAACTATGAAGTCTAAGGCCAACT, R: ATATAAAGACAGCCCGTGGACTTAGG). T7-Kdm4d PCR product was purified and used as the template for in vitro transcription (IVT) using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, AM1345). The RNA products were purified using MEGA clear kit (Life Technologies, AM1908) and eluted in RNase-free water.

Immunostaining Monkey SCNT embryos (one-cell stage) were fixed in PBS with 4% paraformaldehyde (PFA) for 20 min. After being washed three times with PBS, embryos were permeabilized with 0.5% Triton X-100 in PBS (permeabilization buffer) for 30 min. After being blocked in a permeabilization buffer with 10% donkey serum for 1h, embryos were incubated for overnight at 4°C with anti-H3K9me3 (1/200, Abcam, ab8898), which were diluted in a permeabilization buffer containing 10% donkey serum. After being washed with PBS for 3 times every 10 min, Cy3 AffiniPure-conjugated donkey anti-Rabbit IgG (1:500, Jackson: 711-165-152) for 2 hr at 37°C. After being washed with PBS and counterstain of nucleus chromosome (nuclear DNA) with DAPI (Sigma), embryos were transfer into a spinning disk for confocal microscopic analysis (Olympus Microsystems).