Kannika Siripattarapravat, Boonya Pinmee, Patrick J Venta, Chia-Cheng Chang & Jose B Cibelli. Somatic cell nuclear transfer in zebrafish. Nature Methods advance online publication Published online: 30 August 2009. doi:10.1038/nmeth.1369. Full article available on request.
Abstract: We developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones.
Masahito Tachibana, Michelle Sparman, Hathaitip Sritanaudomchai, Hong Ma, Lisa Clepper, Joy Woodward, Ying Li, Cathy Ramsey, Olena Kolotushkina, Shoukhrat Mitalipov. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature (26 August 2009) doi:10.1038/nature08368. Full article available on request.
Abstract: Mitochondria are found in all eukaryotic cells and contain their own genome (mitochondrial DNA or mtDNA). Unlike the nuclear genome, which is derived from both the egg and sperm at fertilization, the mtDNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin. Mutations in mtDNA contribute to a diverse range of currently incurable human diseases and disorders. To establish preclinical models for new therapeutic approaches, we demonstrate here that the mitochondrial genome can be efficiently replaced in mature non-human primate oocytes (Macaca mulatta) by spindle–chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg. The reconstructed oocytes with the mitochondrial replacement were capable of supporting normal fertilization, embryo development and produced healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infants born so far originated from the spindle donors whereas mtDNA came from the cytoplast donors. No contribution of spindle donor mtDNA was detected in offspring. Spindle replacement is shown here as an efficient protocol replacing the full complement of mitochondria in newly generated embryonic stem cell lines. This approach may offer a reproductive option to prevent mtDNA disease transmission in affected families.
Dr. Thuan, from Konkuk University in Seoul, Korea, states "the XYClone is a very convenient tool and excellent for enucleation of porcine and bovine oocytes for somatic cell nuclear tansfer."
Scientists at Newcastle University have created part-human, part-animal hybrid embryos for the first time in the UK. An unfertilized cows egg was cut open by the XYClone laser, and virtually all the genetic material was extracted. DNA derived from a human skin cell was then injected into the egg. By using electric shock, the hybrid embryos started growing. It grew for 3 days to 32 cells. The embryo is 99.9% human and 0.1% cow. They hope to grow them for 6 days to extract the stem cells.
V.J.Hall, D.Compton, P.Stojkovic, M.Nesbitt, M.Herbert, A.Murdoch and M.Stojkovic Human Reproduction, 2007. Jan. 22(1):52–62.
BACKGROUND: Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes.
METHODS: Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT–PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5].
RESULTS: For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes.
CONCLUSIONS: Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.
Senior Research Associate
University of Newcastle
Institute of Human Genetics
Department of Stem Cell Biology and Developmental Genetics
The XYClone laser has been of huge benefit to developing our technique of human somatic cell nuclear transfer. Prior to using the XYClone we were relying on traditional enucleation procedures to remove the genetic material from human oocytes. This generally requires piercing through the elastic outer protein shell of the egg by using a microscopic bevelled glass pipette, which often induced damage and resulted in lysis of the egg. The elasticity of the human zona pellucida compared with other species remained a challenge. However, following installation of the XYClone laser objective, we have been able to speed the process of enucleation to a matter of seconds, without eliciting damage to the egg. The XYClone laser suitably makes a very small, neat slit in the zona pellucida which faciliates the DNA removal. We have been very impressed by how it has dramatically improved our enucleation efficiency and reduced the lysis rates of the eggs. It has also been incredibly easy to operate and we love how easy it is to target the site of interest using the software provided. It appears to be an extremely powerful tool that has improved our ability to create human cloned embryos for the purposes of deriving patient specific stem cell lines which may one day be used to treat non-curable diseases.