Somatic Cell Nuclear Transfer Procedures Research Articles

63 PRODUCTION OF CLONED TRANSGENIC RED CATS

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Because the feline gene map displays a higher level of systemic conservation with the human genome than with rodents or other laboratory mammals, cats may offer a more appropriate model in which to study potential therapies for human diseases. A second point of application is the production of transgenic cats, genetically engineered designer pets with favorable attributes, such as allergen-free cats. Mature oocytes from superovulated cat ovaries were collected. Donor fibroblasts were obtained from an ear-skin biopsy of a white maleTurkish Angora cat, cultured for one to two passages, and then subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. Single donor cells emitting red fluorescence were selected under an inverted microscope equipped with an RFP filter set. For somatic cell nuclear transfer (SCNT), denuded oocytes were treated with 0.2 µg m L–1 demecolcine for 1 h, and the protruding first polar body and chromatin plate were removed by micromanipulation. A single donor cell was transferred into each enucleated oocyte. In total, 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 � 7.5 per recipient). Three of eleven surrogate mothers (S) were successfully impregnated (27.3%). Surrogate S-2 delivered one liveborn and one stillborn kitten at 65 days of gestation while S-3 delivered one liveborn kitten at 66 days of gestation. Microsatellite analysis revealed that all kittens were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cats, but not in the non-TG cats under excitation emission filter sets. The expression pattern of the RFP transgene was determined in several tissues taken from TG stillborn and non-TG cats. Although expression in the heart was detected more weakly than in other organs, red fluorescence was present in all tissue samples tested, but not in those from the non-TG cat. This is the first report of the successful production of a cloned transgenic cat expressing an exogenous gene. Our nuclear transfer procedure using genetically modified somatic cells would be useful for the efficient production of transgenic cats. The application of the SCNT procedure to produce genetically modified cats would be valuable for biomedical modeling of human disease, as well as for the production of designer pets. This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MOST) (grant # M10525010001-05N2501-00110).

Bioethical aspects of regenerative and reproductive medicine

The birth announced in 1997 of Dolly, the lamb cloned from the somatic mammary cells of an adult ewe, and the discovery of human embryonic stem cells in 1998 have been the most exciting developments in the biological sciences in the past decade. Reproductive somatic cell nuclear transfer (SCNT) in additional species has been inefficient in that relatively few births, harmful side effects and high fetal and neonatal death rates have resulted from many attempts. Ongoing debates about the ethics of reproductive SCNT have revealed that some researchers regard human reproductive SCNT as morally unacceptable in all circumstances, others see merit in reproductive SCNT in certain circumstances and others await more information before making judgment about the ethical status of the procedure. Regenerative medicine and emerging biotechnologies started to revolutionize the practice of medicine. Advances in stem cell biology, including embryonic and postnatal somatic stem cells, have made the prospect of tissue regeneration a potential reality. Mammal cloning experiments have provided new impetus to the prospect of regenerative medicine through stem cell research. The procedure of SCNT could be used to create the raw material to replace defective or senescent tissue as a natural extension of the biology of stem cells. Researchers working in reproductive medicine should consider the potential hope given to many patients against the requisite and ethically contentious creation of human blastocysts for therapeutic intent.

94 PRODUCTION OF AN α1,3-GALACTOSYLTRANSFERASE GENE KNOCKOUT CALF USING A SINGLE TARGETING VECTOR AND REJUVENATED CELL LINES

Gene targeting in large animals has the potential to be useful in agriculture. In this study, we examined whether an �1,3-galactosyltransferase-gene knockout (null) calf could be produced using a single targeting vector for disruption of both alleles and a rejuvenation of cell line by production of cloned fetuses. A promoter-less positive selection vector (pGT-22) containing an IRES (internal ribosome entry site)-antibiotic resistance gene(neo) cassette and loxP sequences was used to disrupt the bovine �1,3-galactosyltransferase (�1,3-GT) gene. In gene-targeting (GT), Japanese Black fetal fibroblasts were transfected with pGT-22, and were selected with 0.4 mg mL-1 G418. G418-resistant cells were monitored by PCR and Southern blot analysis. After PCR selection, a portion of the PCR-positive colonies were infected with the adenovirus AxCANCre, which transiently expresses Cre recombinase in the infected cells, to excise the antibiotic-resistance gene cassette (IRES/lacZ-neo) from the targeted allele. The targeted cells in which homologous recombination occurred were used for somatic cell nuclear transfer (SCNT). The cell cycle synchronization of donor cells in the early G1 phase and SCNT were performed according to the established procedure in our laboratory (Urakawa et al. 2004 Theriogenology 62, 714–728). As a result, in the first GT, one PCR-Southern blot-positive clone (0.26%, in 380 G418-resistant colony) was used for SCNT. Of 35 pulsed SCNT embryos, 4 (11.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT+/-) was established and was transfected with pGT-22 in the second GT. As a result, 8 PCR-Southern blot-positive clones (1.6%, in 508 G418-resistant colony) were recovered. The 2nd GT and the following SCNT were accomplished by the same methods used in the 1st GT and SCNT procedure. Of 36 pulsed SCNT embryos, 7 (19.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT-/-) was established, and was used for SCNT to obtain the �1,3-GT homozygous knockout offspring. Finally, 52 of 148 pulsed SCNT embryos (35.1%) developed to the blastocyst stage and 6 cloned embryos were transferred into 3 recipient heifers. A single knockout female offspring with normal morphology in all organs was born (Day 287) but died about one hour after birth. In this study, we established both heterozygous and homozygous �1,3-GT knockout cell lines from primary fetal fibroblasts and produced an �1,3-GT knockout offspring using a single targeting vector and rejuvenated cell lines.

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7-19 of gestation.

Analysis of mRNA Transcripts for Insulin-Like Growth Factor Receptors and Binding Proteins in Bovine Embryos Derived from Somatic Cell Nuclear Transfer

The low efficiency of animal production using somatic cell nuclear transfer procedures is considered to be the result of an incomplete reprogramming of donor cell nucleus, which leads to abnormal expression of developmentally important genes. The objective of this study was to determine the abundance of gene transcripts of insulin-like growth factor (IGF)-related genes in cloned bovine embryos reconstructed with somatic cells. Single embryos derived from nuclear transfer reconstructed with somatic cells (NT-SC) or embryo blastomeres (NTEM), in vitro fertilization (IVF), in vivo production (Vivo), and parthenogenetic treatment (PA) were analyzed. The relative abundance of mRNA was examined by real-time PCR. Transcripts of the IGF-1 receptor (r) and IGF binding protein (BP)-2 were detected in all embryos, regardless of origin. IGF-IIr and IGFBP-3 transcripts signals in NT-SC embryos were detected with significantly lower frequencies of 25 and 50%, respectively. Although IGF-Ir and IGFIIr transcript levels were not significantly different in NT-SC, NT-EM, IVF, Vivo, and PA embryos, the relative abundance in individual embryos indicated large variation in NT-SC. IGFBP-2 and IGFBP-3 levels were high in the Vivo embryos compared with NT-SC, NT-EM, IVF, or PA embryos. These results suggest differences in levels of transcripts of IGF-related genes in the bovine embryos produced by NT compared with IVF, Vivo, and PA.

75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.

Development of bioassays using the bovine model to measure the efficiency of SCNT in humans

Development of bioassays using the bovine model to measure the efficiency of SCNT in humans

Stem Cell Research: Why Medicine Should Reject Human Cloning

Stem Cell Research: Why Medicine Should Reject Human Cloning

Analysis of Gene Transcription in Bovine Nuclear Transfer Embryos Reconstructed with Granulosa Cell Nuclei1

The low efficiency of animal production using somatic cell nuclear transfer procedures is considered to be the result of an incomplete reprogramming of the donor somatic cell nucleus, which leads to a lack of, or abnormal expression of developmentally important genes. However, our current understanding of the process of somatic cell nuclear reprogramming and its effect on gene expression is limited. In this study, we compare the transcription patterns of six developmentally important genes, Oct4, IL6, FGF2, FGF4, FGFr2, and gp130 in single in vitro fertilized (IVF) and nuclear transfer embryos reconstructed using granulosa cells for the donor nuclei. Similar patterns of transcription were detected for Oct4, FGF2, and gp130 in IVF and nuclear transfer embryos during the preimplantation stages of development. However, a number of morula- and blastocyst-stage embryos derived from nuclear transfer procedures showed abnormal transcription of IL6, FGF4, and FGFr2. Previous studies have demonstrated that these three genes play an important role in implantation, early postimplantation development, or both in the mouse. Therefore, the aberrant transcription patterns detected in nuclear transfer embryos may lead to a reduction in embryo viability.

Reprogramming cattle somatic cells by isolated nuclear injection.

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts). The use of double cytoplasts did not improve cleavage, and development to blastocysts could not be achieved. In a comparison of electrofusion of fibroblasts with cytoplasts in the subzonal perivitelline space with intracytoplasmic injection of nuclei and parthenogenetically activated oocytes, 2%, 14% and 24% developed to blastocysts respectively. In the group injected with isolated nuclei, the passage number (4 to 9) had no apparent influence on developmental competence to blastocysts. The embryos produced by nuclear injection of somatic cell nuclei showed the normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific epitopes during development, as seen in fertilized oocytes. We conclude that the nuclear injection of somatic cell nuclei is a relatively efficient way to clone bovine embryos.