SUPPORTING ONLINE MATERIAL

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1 SUPPORTING ONLINE MATERIAL SUPPORTING ONLINE TEXT Efficiency of SCNT Alive fetuses at mid-gestation The rate of viable (beating heart) embryos at day dpc was assessed after sacrifice of foster mothers and immediate dissection of implantation sites under stereoscopic magnification. Reimplantation of 876 embryos in 12 recipients lead to the obtention of 13 (1.5%) alive fetuses. Alive fetuses at term Pups originating from XY fibroblasts 129 reconstructed oocytes were reimplanted into 2 fosters. Three (2.3%) alive male pups were recovered from one recipient but one died within a few hours. Pups originating from XX fibroblasts Two female recipients transfered with 12 reconstructed embryos died during anaesthesia. Part of the transfered cloned embryos could have been rapidly recovered by flushing out from the oviducts. Unfortunately, lack of counting of the recovered embryos before their subsequent transfer into a third available recipient precluded accurate calculation of success rate for these embryos. At final, two female clones were obtained from this foster mother.

2 SUPPORTING ONLINE MATERIALS AND METHODS 1. Cells and media Primary rat embryonic fibroblasts were isolated from CD-SpragueDawley fetuses obtained from females at day 12.5 of pregnancy according to the same protocol as described for mouse fetal fibroblasts (S1). Cells were used for nuclear transfer from passage 1 to passage 3. Fibroblasts were synchronized in metaphase according to the protocol slightly modified of Zhou et al. (S2). Confluent cultures were passaged the day before and diluted 4 times. On the morning of nuclear transfer Demecolcin (Sigma Chemical Co., St. Louis, MO) was added to fibroblasts dishes (final concentration:.5 µg/ml). After 2 hours of incubation, the mitotic cells loosely attached to the dish were recovered in the medium after shaking the flasks. Cells were washed twice in the medium free of drug and were finally resuspended in 2µl of M2 medium. Handling of oocytes and embryos outside the incubator was performed in M2 (Sigma) medium while in vitro culture of reconstructed oocytes and embryos was achieved in microdrops of 5µl of rat embryo culture medium (R1ECM) (S3) containing 11µM NaCl under oil at 37 C, 5% CO2 / air. 2. Standard oocytes collection and handling Three to 4 weeks old OFA-SpragueDawley females were superovulated by intraperitoneal injections of ecg (1IU) and hcg (1IU) given 48 to 52h apart. Oocytes were recovered 14h after hcg injection and were processed for cumulus removal in prewarmed (37 C) R1ECM containing 33mg/ml hyaluronidase. Denuded oocytes were then rinsed in M2

3 medium and immediately transferred to a 5µl of the same medium drop under oil for nuclear transfer. 3. Control of rat oocyte activation General protocol followed for the application of drugs acting on oocyte activation in SCNT procedure is presented in Fig. S1. Inhibition of spontaneous activation before SCNT Prewarmed (37 C) M2 medium containing 5µM of MG132 (VWR, France) was used throughout egg collection. Cumulus were recovered from oviducts immediately after female sacrifice and dissociated in a 5µl drop of medium containing 33mg/ml hyaluronidase. Denuded oocytes were then rinsed in the medium free of hyaluronidase and used immediately for nuclear transfer. Induction of activation after SCNT Activation was induced by transfer of reconstructed oocytes to R1ECM containing 15µM Butyrolactone I (Affiniti R.P.L, Mamhead, UK) for a 2h incubation. Butyrolactone allowed expulsion of polar body, formation of normal appearing pronuclei and occurence of first mitotic cleavage in the majority (9%) of reconstructed oocytes. In the absence of Butyrolactone reconstructed oocytes could activate spontaneously in vitro in the free-mg132 culture medium but this process was restricted to only expulsion of polar body. 4. Nuclear Transfer The SCNT procedure was performed at RT in MG132-free M2 medium. A fibroblast cell was aspirated into a 1µm I.D pipette resulting in membrane cell lysis and was injected

4 into oocytes at the opposite side of the MII metaphase location after zona drilling and oolemma breaking using piezo actuation. The injection pipette was then slowly withdrawn until being close to oocyte metaphase chromosomes. Oocyte's chromosomes were then sucked into the pipette allowing closure of oocyte's oolema. 5. In vitro culture of embryos After exposure to butyrolactone, reconstructed embryos were washed several times in drug free R1ECM medium and transfered in drops of the same medium for overnight culture until reimplantation into foster mothers. 6. Embryo transfer Two-cell stage embryos were transferred to the oviduct of OFA-SpragueDawley.5 dpc pseudopregnant females (mating the night before the reimplantation with vasectomized males of the same strain and of proven sterility). 7. DNA analysis of oocytes Oocytes were fixed in 2% paraformaldehyde (PFA) in PBS for 2 min, permeabilised with.2% Triton X-1 for 15 min, rinsed in PBS, and labeled by addition of 1 mg/ml propidium iodide for 3 min (all steps were performed at RT). Oocytes were then mounted on slides with the antifading agent Vectashield (Vector) and observed under a confocal laser scanning microscope (Carl Zeiss, CLSM 31). 8. Genotyping of live rats obtained after somatic cell nuclear transfer Genotyping analysis was performed on cloned rats and on control rats of OFA-SD (n=46) and of CD-SpragueDawley (CD-SD) (n=45) origin. Tong biopsies were collected in the two production colonies of Charles River Laboratories France where all animals used for the SCNT experiments were produced: oocyte producers

5 (OFA-SpragueDawley: OFA-SD), vasectomized rats (OFA-SD), fosters (OFA-SD) and embryos for fibroblast production (CD-SD). Total genomic DNA was prepared following standard procedures from rat tongs using a Biorad kit (ref : ). Genetic status of rats was assessed by PCR amplification of 2 satellite markers, namely D1Wox22 in the Igf2 gene and D2Wox17 in the Tyr1 gene (table 1) (Bihoreau et al. 1997). PCR primers for microsatellite markers were labelled with a fluorescent dye (6-FAM). Genotypes were determined by capillaries gel electrophoresis using the ABI31 DNA sequencer and were analyzed by the Genscan and Genotyper softwares (Applied Biosystems PerkinElmer). a) Genotypes of OFA-SD and CD-SD populations The data on genotyping analysis and related allele frequencies are summarized in tables 2 and 3, respectively. Microsatelitte analysis in the Igf2 gene The 13 bp Igf2 allele is homozygous in all animals of the OFA-SD population (46 out of 46 colony male reproducers) whereas three alleles 13, 132 and 14 bp are detected in the CD- SD population with a frequency of 5.%, 44.4% and 5.6%, respectively. Our results are similar to those courteously provided by Charles River Laboratories (P. Hardy, Scientific Director, Charles River Laboratories France, pers. comm.) with only one Igf2 allele (13bp - D1Wox22 microsatelitte) being present in OFA-SD rat colony (n=96). Microsatelitte analysis in the Tyr1 gene In OFA-SD, the 174 bp Tyr1 allele represents 94.6% of the allele frequency and the 165bp allele the remaining 5.4%, whereas in CD-SD the alleles 165, 167 and 174 bp have a frequency of 58.9%, 7.8% and 33.3%, respectively. Our results are similar to those courteously provided by Charles River Laboratories (P. Hardy, Scientific Director, Charles River Laboratories France, pers. comm.) with the 174 bp

6 Tyr1 allele (D1Wox17 microsatelitte) representing 95% of the allele frequency in OFA-SD rat colony (n=96). b) Genotypes of cloned rats Data presented here relate to the 4 live animals (2 males and 2 females) obtained after SCNT using fibroblasts of CD-SD genetic background as somatic nucleus donor cells. The data on genotyping analysis and related allele frequencies are summarized in tables 2 and 3, respectively. Statistics Probabilities that cloned animals were of CD-SD ( ) or OFA-SD (ph 1 ) genotype were calculated for each marker, for both markers and then compared to each other ( / ph 1 ). The computation was performed on the data obtained from our genotyping experiment only. The data provided by Charles River Laboratories were not included. Igf2 marker = 22/45 x (2/45) 4 x 24 = ph 1 * = (1/48) 4 x 24 = / ph 1 = Try1 marker = (18/45) 4 x 24 = ph 1 = (5/46) 4 x 24 = / ph 1 = Igf2 and Try1 markers / ph 1 = x = * The frequency of the 13/132 and 132/14 genotypes was estimated to be 1/48 in the OFA- SD genetic background although overestimated according to the CR laboratories data.

7 b) Conclusion The estimated probability that the obtained animals were of CD-SD genetic background and therefore derived from fibroblast nuclei after nuclear transfer is about 4, higher than the probability to be of OFA-SD genetic origin resulting from sperm fertilisation. This conclusion is strongly supported by the genotyping results obtained at Charles River Laboratories. We conclude that these rats derived from somatic cell nuclear transfer. References S1. B. Hogan et al. "Manipulating the Mouse Embryo" A laboratory manual, Second Edition, Cold Spring Harbour Laboratory Press (1994). S2. Q. Zhou et al., Biol. Reprod. 65, 412 (21) S3. K. Miyoshi et al., Biol. Reprod. 56, 18 (1997) S4. M.T. Bihoreau et al., Genome Res. 7, 434 (1997)

8 Fig.S1 Control of rat oocyte activation in somatic cell nuclear transfer. Oocytes are maintained at MII stage until somatic cell nuclear transfer (SCNT) by incubation in MG132 supplemented medium. SCNT is performed in MG132 free medium. Activation is initiated by transfer of reconstructed oocytes into Butyrolactone containing medium. MG132 Inhibition of spontaneous activation BLI Induction of activation MII Oocyte Nuclear Transfer in drug free medium Foster mother Embryonic developmen t Nucleus donor cells Cloned rat

9 Table S1. satellite markers used Marker Gene sequence (chromosome) D1Wox22 Igf2 (1) TAC CCA CAC GTA CAT GCA CA CAA TGT GGT TCC AAT CGA AG D4Wox24 Try (1) ACC CTA AGG CTC TGT CTC AAA CTT TGG ATA GTA ATG AGT GCT TTG Table S2. population per genotype Igf2 CD-SD OFA-SD clones 13/13* /132* 8 13/ ** 13/14* 132/ *** Try1 165/165* 174/174* 165/167* 165/ /174 * PCR fragment size ** female *** 2 males and 1 female Table S3. Allele frequency CD-SD OFA-SD clones Igf Igf Igf Try Try Try