Donor Cells For Somatic Nuclear Transfer Research Articles

Comparative Potential of Cultured Skin Fibroblast, Cumulus, and Granulosa Cell to Produce Somatic Cell Nuclear Transfer (SCNT) Preimplantation Embryos in Buffaloes (Bubalus bubalis) in Relation to Gene Expressions

To improve the efficiency of somatic cell nuclear transfer (SCNT)-derived embryos in buffaloes (Bubalus bubalis), skin fibroblast, cumulus, and granulosa cells were cultured up to the 15th passage and cloned embryos were produced from each cell type. At the 15th passage the cumulative population doublings (CPDs) in cumulus cells was higher (60.78) than skin fibroblast (57.12) and granulosa (56.05) cell lines. Gene expression of chromatin remodelling proteins, that is, HDAC1, DNMT1, DNMT3a, and DNMT3b, were comparable at all five passages (P-3, P-6, P-9, P-12, and P-15) groups in cumulus cells but different in skin fibroblast and granulosa cells. Cleavage and blastocyst production rate in cumulus (65.9 and 27.4%)-derived embryos was higher than skin fibroblast (63.8 and 24.3%) and granulosa (62.5 and 22.3%)-derived embryos. Expressions of HDAC1, DNMT1, and DNMT3a mRNA in cumulus-derived blastocysts were similar to IVF blastocysts (control), whereas skin fibroblast and granulosa-derived blastocysts expression was significantly different (p < or = 0.05). DNMT3b mRNA expression in all the three donor cell types and IVF control were similar. The expression pattern of these genes showed the effect of donor cell type with different epigenetic reprogramming capabilities for SCNT embryo production rate. Overall, results indicated that cumulus cells are the best nuclear donor for SCNT.

The influence of donor nucleus source on the outcome of zebrafish somatic cell nuclear transfer

The success of nuclear reprogramming following somatic cell nuclear transfer (SCNT) is thought to depend on factors present in the egg. Little is known about the role - if any - played by the somatic cell type on the outcome of the procedure. We tested whether cells of different lineages might have different capacities for reprogramming following SCNT, comparing cells isolated from five different tissues of transgenic zebrafish for their developmental potential when used as SCNT donor cells. We used transgenic zebrafish lines expressing green fluorescence protein under an endogenous tissue-specific promoter: HGn62A-skin, HGn28A-skin, HGn8E-heart, HG21C-fin and notochord and HGn30A-hatch gland. We analyzed the efficiency of cloning, as measured by reconstructed embryos that developed up to the hatched-fry stage. Specifically, donor cells of fin and notochord origin yielded the best rate of cloned fish production. All of the other cell types used were capable of producing cloned fish, albeit with significantly lower efficiency. These results indicate that the type of zebrafish cells used for SCNT can influence the outcome of the procedure. Future epigenetic analysis of these cells will help determine specific chromatin profiles in somatic cells that have an impact on nuclear reprogramming procedures.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Efficiency of human lactoferrin transgenic donor cell preparation for SCNT

The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5 ± 2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.

Correlation of Developmental Differences of Nuclear Transfer Embryos Cells to the Methylation Profiles of Nuclear Transfer Donor Cells in Swine

Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues. The methylation profiles of the donor cells were then analyzed with respect to their ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2, A7, and A8 had blastocyst rates of 11.7%a, 16.7%ab, and 20.0%b, respectively (ab P

40 EARLY DEVELOPMENT OF RECONSTRUCTED MOUSE EMBRYOS USING BONE MARROW CELLS FROZEN WITHOUT CRYOPROTECTANT

Animal cells frozen with suitable cryoprotectants have been successfully cryopreserved for long periods of time, maintaining viability upon thawing. Animal cells frozen without cryoprotectant, however, may suffer serious damage and not be useful as donors in somatic cell nuclear transfer (SCNT). However, in some cases, old animal samples were frozen only as a whole body or a piece of tissue without cryoprotectant. If the cells from such old samples could be useful for SCNT, then there are potentially many candidates where individual animals could be reproduced. In this study, we examined the possibility of using mouse bone marrow cells frozen without cryoprotectant as nuclear donors in SCNT. Thigh bones were collected from B6C3F1 mice and frozen in either a –25�C or a –80�C freezer for more than one month. Thawing of frozen bones was performed by placing them in an incubator at 37�C. Bone marrow cells were collected by washing the bone cavity with saline. Recipient oocytes for SCNT were collected from B6D2F1 female mice. The enucleation of recipient oocytes and the injection of nuclei were performed as previously reported (Wakayama et al. 1998 Nature 394, 369–374) with a piezo-actuated micromanipulator system. In this study, 4 groups of mouse cells (fresh bone marrow cells, bone marrow cells frozen at –25�C, bone marrow cells frozen at –80�C, and fresh cumulus cells) were used as the nuclear donors in SCNT. After nuclear injection, embryos were kept in mCZB medium for 1 h at 37�C. Subsequently, embryos were cultured for 3 h with 5 µg mL–1 cytochalasin B and 10 mm SrCl2 for activation and cultured for an additional 20 h in mKSOM medium. The nuclear dynamics of SCNT embryos in each donor cell group was observed using 42,6-diamidino-2-phenylindole (DAPI) staining and a fluorescent microscope at 0, 1, 7, and 24 h after nuclear injection. Data were analyzed by Student's t-test. The cell viability after thawing by trypan blue vital staining was about 20% regardless of freezing temperature. At 7 h after nuclear injection, the SCNT embryos injected with frozen bone marrow cells, regardless of freezing temperature, had more single pronuclei (67%, 54/81; P &lt; 0.05) than SCNT embryos injected with either fresh bone marrow cells (36%, 26/73) or cumulus cells (28%, 67/236). At 24 h after nuclear injection, fewer SCNT embryos injected with bone marrow cells, either fresh or frozen, developed to the 2-cell stage (fresh: 11%, 6/56; frozen at –25�C: 21%, 5/24; frozen at –80�C: 20%, 10/49) than SCNT embryos injected with cumulus cells (58%, 185/319; P &lt; 0.05). There was no difference in the embryonic development to the 2-cell stage among SCNT embryos injected with either fresh or frozen bone marrow cells. Further studies are required to determine whether cells frozen without cryoprotectant are capable of resulting in viable clones.

54THE DEVELOPMENT AND CHARACTERIZATION OF MITOCHONDRIAL DNA (MTDNA)-DEPLETED CAPRA HIRCUS FETAL FIBROBLASTS: CANDIDATE DONORS FOR SOMATIC CELL NUCLEAR TRANSFER (SCNT)

Mammalian mtDNA is approximately 16.6kb in size. It has 37 genes, 13 of which encode protein subunits of the oxidative phosphorylation (OXPHOS) system, the major ATP-generating pathway of the cell. Normally, mammals inherit a single mtDNA genome (homoplasmy) from their mother. Somatic cell nuclear transfer (SCNT) violates this strict maternal, homoplasmic inheritance of mtDNA as cytoplasm is transferred along with the nucleus, which often results in an oocyte harboring both donor and recipient mtDNA genomes (heteroplasmy). This been previously reported (reviewed St. John JC 2002 Theriogenology 57, 109–123). To overcome the problem of donor mtDNA transmission, we have developed and characterized mtDNA-depleted C. hircus (goat) cells for use as donors in SCNT. C. hircus primary foetal fibroblast cells were established in culture and depleted of their mtDNA by supplementing their growth medium with a low concentration, 50ngmL−1, of ethidium bromide (EthBr). Conventional PCR, using a series of primers designed specifically for goat mtDNA, was used to screen for the presence of mtDNA during the EthBr treatment. In addition, mitochondrial organization, activity and morphology in the cells were analyzed using the mitochondrial specific fluoroprobe JC1. mtDNA-encoded and mitochondrial transcription factor A (mtTFA) transcript levels were analysed using RTPCR. Furthermore, both mtDNA depleted and non-depleted cells were characterised using immunocytochemistry to detect the expression of specific protein subunits of the OXPHOS system. Progressive mtDNA depletion was observed, using conventional PCR, in cells treated for 3 to 25 days with EthBr, while 42 days of culture resulted in complete depletion. RTPCR showed a progressive reduction followed by complete elimination of the mtDNA-encoded ND1, ND2, ND3, COX I and mtTFA transcripts. In addition, the expression of mtDNA-encoded protein subunits, e.g. COXI, of the OXPHOS system were reduced following mtDNA depletion whereas the expression of nuclear-DNA encoded protein subunits, e.g. COXVic, were unaltered. We hypothesize that the elimination of mtDNA and mtDNA transcripts from the donor cells will facilitate normal mtDNA replication and transcription in SCNT embryos, thus maintaining the strict unimaternal transmission of mtDNA to the offspring. Consequently, genetically identical offspring will be generated which have identical nuclear and mitochondrial DNA content, assuming oocytes from the same ovary are used. This technique is important for the generation of offspring for the livestock industry and animal models for the analysis of single gene disorders as well as the propagation of endangered species.