Comparison of Microinjection (Piezo-Electric) and Cell Fusion for Nuclear Transfer Success with Different Cell Types in Cattle

Abstract

Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.

The effect of mitochondrial genome on architectural remodeling and epigenetic reprogramming of donor cell nuclei in mammalian nuclear transfer-derived embryos

“…In this case the only source of exogenous proteins and mRNA transcripts is the nucleoplasm of transplanted karyoplast. Insignificant amounts of perinuclear cytoplasm (perikaryon) remain to interfere with the further embryonic development of mammalian clonal zygotes (Lacham-Kaplan et al, 2000;Prather, 2000;Galli et al, 2002;Roh and Hwang, 2002;Lee et al, 2003c;Samiec, 2004;St. John et al, 2004;Yang et al, 2004).…”

The role of mitochondrial genome (mtDNA) in somatic and embryo cloning of mammals. A review

“…Also used frequently are the procedures of microsurgical nuclear transfer, involving both direct karyoplast (cell nucleus with residual, perinuclear protoplasmic "ring" i.e. perikaryon) injection into the cytoplasm of an enucleated oocyte (pigs: Onishi et al, 2000, Roh and Hwang, 2002, Samiec et al, 2003a,b, Skrzyszowska et al, 2003, Skrzyszowska and Samiec, 2004mice: Wakayama et al, 1998cattle: LachamKaplan et al, 2000, Do et al, 2002, Galli et al, 2002horses: Choi et al, 2002), and whole cell intraooplasmic microinjection (pigs: Lee et al, 2003b). However, in the nuclear transfer technique by somatic cell-cytoplast couplet electrofusion or by whole cell intracytoplasmic microinjection, heteroplasmic sources of mtDNA from a donor cell and an enucleated recipient oocyte are mixed in the cytoplasm of the reconstituted embryo ( Figure 1).…”

Development of pig cloning studies: past, present and future

“…Also important are the efficacies of the exogenous somatic nuclei transplantation procedure for maternal chromosome elimination (recipient oocyte enucleation), the oocyte reconstruction technique and the clonal cybrid (cytoplasmic hybrid) activation system (Kato et al, 2000;Polejaeva et al, 2000;Wakayama and Yanagimachi, 2001;Galli et al, 2002;Roh and Hwang, 2002;Yin et al, 2002;Nagashima et al, 2003;Samiec et al, 2003a,b). At present, the most common embryo reconstruction techniques are nuclear transfer followed by electric pulse fusion of cytoplast (ooplast) with somatic cell (cattle: Cibelli et al, 1998;Wells et al, 1999;Kato et al, 2000;sheep: Wilmut et al, 1997;McCreath et al, 2000;goats: Baguisi et al, 1999;pigs: Koo et al, 2000;Polejaeva et al, 2000;Boquest et al, 2002;Yin et al, 2002) or the use of a suspension of inactivated Sendai virus (HVJ) particles with an accurately defined number of haemagglutination activity units (mice: Kato et al, 1999;Ono et al, 2001).…”

“…The procedures of microsurgical nuclear transfer, involving both direct karyoplast (cell nucleus with residual, perinuclear protoplasmic "ring" i.e. perikaryon) injection into the cytoplasm of an enucleated oocyte (pigs: Kuhholzer et al, 2000;Onishi et al, 2000;Samiec et al, 2003a,b;Roh and Hwang, 2002;mice: Wakayama et al, 1998;Chung et al, 2002;cattle: Lacham-Kaplan et al, 2000;Galli et al, 2002;horses: Choi et al, 2002), and whole cell intraooplasmic microinjection (pigs: Lee et al, 2003b) are also used frequently.…”

“…On the other hand, in the studies carried out by Nagashima et al (2003), using foetal fibroblast cell nuclei simultaneous comparison between two nuclear transfer methods (ooplast-donor cell complex electrofusion and intracytoplasmic piezo-electric microinjection into enucleated oocytes) revealed clear differences in the pattern of nuclear remodeling and developmental potential of embryos to blastocyst stage as well as in the morphological quality of the clonal blastocysts as measured by total cell number. However, considering the results of the majority of the studies for nuclear transfer in farmed livestock species (Lacham-Kaplan et al, 2000;Ogura et al, 2000;Park et al, 2001a;Wakayama and Yanagimachi, 2001;Galli et al, 2002), it can be concluded that intraooplasmic injection of karyoplasts prepared from cells at G0/G1 or G2/M stages of cell cycle could also increase considerably the total efficiency of somatic cloning in pigs and other mammalian species.…”