47 BLASTOCYST DEVELOPMENT RATE OF CLONED BOVINE EMBRYOS USING SERIAL NUCLEAR TRANSFER OF CELLS CONTAINING AN X-AUTOSOME-TRANSLOCATED CHROMOSOME t(Xp+;23q-)

Somatic cell nuclear transfer (SCNT) has been utilized to study various genetic and epigenetic contributions of specific biomedical diseases and developmental events by using various donor cell types such as mature lymphocytes, brain tumor cells, and other unique genotypes. Previously, we produced cloned fetuses and offspring derived from SCNT of adult ear skin fibroblasts obtained from a sub-fertile cow harboring an X-autosome translocation as a model to study X-inactivation and chromosome dynamics during female meiosis. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned calf with the X-autosome translocation t(Xp+;23q-) compared to the original adult fibroblast donor containing the same chromosome translocation. Primary cultures of cells were established in DMEM +15% fetal calf serum (FCS). To serve as nuclear donors, cells at 5-7 passages were cultured for 5 days until confluent. Oocytes matured for 18 h in TCM-199 with hormones were removed of their chromatin, and reconstructed by transfer of donor cells and fusion with two DC pulses (1.2 kV/cm, 15 �s), delivered by a BTX 2000 Electro Cell Minupulator (BTX, Inc., San Diego, CA, USA), in 0.28 M mannitol containing 0.01 mM MgCl2. After 1 h of fusion, the eggs were activated with 5.5 �M ionomycin for 5 min, followed by 11 �g/mL cyclohexamide for 5 h. The eggs were cultured for 9 days in L-SOF at 39�C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2. Chi-square analysis revealed no significant (P > 0.05) differences in the rates of cleavage, blastocyst frequencies, and cell numbers between the 1st and 2nd generation cloned embryos. Cleavage rates were 87.4% and 85.4% for 1st and 2nd generation cloned embryos, respectively. The frequencies of blastocyst development and hatched blastocyst formation on Day 9 were 41.4% (91/220) and 38.7% (92/238), and 26.4% (58/220) and 22.7% (54/238) for the 1st and 2nd generation cloned embryos, respectively. The numbers of total cells and inner cell mass (ICM) cells of Day 9 blastocysts were 183 and 52, respectively, in the 1st generation embryos and 167 and 51 cells in the 2nd-generation cloned embryos. In summary, 2nd generation cloned embryos derived from fibroblasts of a cloned calf with an X-autosome translocated chromosome showed embryo development and cell numbers similar to those of the 1st generation clones. These results demonstrate that serial nuclear transfer does not improve the blastocyst development rate of cloned embryos containing the X-autosome translocation t(Xp+;23q-). This work was funded by OCAG, OMAF, and CRC.

Production of cloned mice from embryonic stem cells arrested at metaphase

In mammals, cloned individuals can be produced from somatic cells. The combined use of gene targeting in embryonic stem cells and cloning contributes to the investigation of gene function in mammals. However, one of the major limitations to cloning is the low viability of cloned embryos, leading typically to high rates of pre- and postnatal death. The present study investigated whether cloning efficiency is influenced by the procedural differences involved in using transfected embryonic stem cells arrested at M phase for cloning by both single and serial transfer. In contrast to a previous study, in which fibroblasts were used, in the present study using embryonic stem cells there was no difference in the rate of production of cloned pups after the use of a single or serial nuclear transfer, although the proportion of blastocysts (70% versus 51%) was significantly higher (P < 0.001) after serial nuclear transfer. After embryo transfer of 445 blastocysts, 218 (49%) implanted and 27 (6% of blastocysts transferred) live pups were born. Of these 27 pups, 23 developed to adults of apparently normal fertility. Of these adults, 39% (n = 9) were derived from targeted embryonic stem cells, which is similar to the proportion of targeted embryonic stem cells in the population used for cloning. This study showed that cloning with embryonic stem cells is a viable procedure resulting in the production of transgenic cloned adults.

Novel method for demonstrating nuclear contribution in mouse nuclear transfer

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.