Ian M. Lewis

Somatic cell cloning without micromanipulators.

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively. This simple, rapid, and inexpensive procedure may become a useful alternative to the existing techniques for somatic cell nuclear transfer for large-scale application of the technology.

The effect of recipient oocyte volume on nuclear transfer in cattle

This study compared the developmental potential of bovine nuclear transfer embryos with varying amounts of cytoplasm. Embryos formed from single cytoplasts fused to blastomeres by a single electrical pulse or from double cytoplasts using a double electrical pulse resulted in reconstituted embryos containing 75% and 150% of the original oocyte volume. No differences in fusion, cleavage, or development rates to blastocysts were observed between the groups. Mean cell numbers 2 days after fusion were significantly lower in single‐cytoplast clones. Cell numbers of resulting blastocysts were likewise significantly lower in single‐cytoplast clones. Embryos formed by fusion of blastomeres with single cytoplasts using a single electrical pulse or from double cytoplasts using either a single or a double pulse resulted in reconstituted embryos containing 50%, 100% and 100% of the original oocyte volume. Again, no differences in fusion or cleavage rates were observed between groups, but the development to blastocysts at day 7 was significantly higher in double cytoplasts constructed with one fusion pulse than in single cytoplasts (P< 0.05). Mean cell numbers 2 days after fusion were significantly lower in single‐cytoplast clones (P< 0.05), but at the blastocyst stage, no statistically significant differences in cell numbers were observed.

Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.

Abstract Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

Comparison of two approaches to nuclear transfer in the bovine : hand-made cloning with modifications and the conventional nuclear transfer technique

The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.

Development of bovine embryo‐derived clones after increasing rounds of nuclear recycling

This study assessed in vitro and in vivo developmental ability of bovine embryo‐derived clones after one, four or seven rounds of nuclear transfer. Initial donor embryo production and all subsequent cultures were performed in vitro. Donor clonal embryo lines were vitrified and warmed either once (first generation), twice (third generation) or three times (sixth generation) before the final round of cloning. No differences were observed in fusion, cleavage and development rates to the 16‐cell stage between the first six cloning generations. Likewise, neither the fusion nor cleavage rates were different between first, fourth and seventh generation clones. However, development to morulae and blastocysts decreased significantly as the number of recycling rounds increased (24.8, 15.1 and 13.6% for first, fourth and seventh generation, respectively). In addition, the proportion of blastocysts compared to morulae decreased, indicating slower developmental speed in later generation clones. After transfer of 16, 25 and 7 clones to 7, 11 and 2 recipients (first, fourth and seventh generation, respectively) initial pregnancy rates of 57, 27 and 0% were obtained. Final rates of calves to term were 25 and 4% per transferred clone for first and fourth generation clones, respectively. These results indicate greatly reduced in vitro and in vivo developmental capacity of bovine embryo‐derived clones after several rounds of nuclear recycling. Whether it is caused by intrinsic factors associated with the genome modification and reprogramming as such, or by external factors such as prolonged in vitro culture period or the effects of vitrification, remains to be determined. Mol. Reprod. Dev. 58:384–389, 2001.

Developmental competence of nuclear transfer cow oocytes after direct injection of fetal fibroblast nuclei.

We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.

Inheritance of Sperm Centrioles and Centrosomes in Bovine Embryos

Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles of slaughtered cow and matured for 24 h in TCM 199 medium with hormones. Eighty-five percent of oocytes matured with subsequent abstriction of the polar body. Matured COCs were then inseminated with frozen-thawed semen (2 x 10(6)/mL final concentration). Eighteen hours after insemination, fertilized COCs were vortexed, washed, and cultured to the pronuclear stage and syngamy (24-36 h postinsemination) and fixed for TEM. Unfixed embryos achieved a cleavage rate of 54%, with 29% developing to blastocysts. Fertilization was confirmed by TEM. Examination of fertilized bipronuclear ova revealed the presence of a sperm aster associated with sperm midpieces, tails, and male pronuclei in several embryos. Further examination of embryos at syngamy showed centrioles at one pole of the first mitotic bipolar spindle in two embryos. Since the mature oocyte at metaphase II has no centrioles at spindly poles, this centriole was most likely derived from the sperm, which has a single proximal centriole associated with pericentriolar material in its neck region, like most mammalian sperm. Tripronuclear ova produced disorganized bipolar spindles or, rarely, tripolar spindles. Bovine embryos, too, follow Boveris rule of paternal inheritance as in man and most animals. It is possible that both paternal centrosomes (centrioles) and maternal centrosomes are involved in the organization of bipolar spindles in these embryos, quite unlike the mouse embryo where maternal centrosomes seem to organize the first mitotic spindle. The bovine embryo appears to be an appropriate model to study centriolar inheritance.

Handmade Somatic Cell Cloning in Cattle

Apart from the biological and ethical problems, technical difficulties also hamper the improvement and widespread application of somatic cell nuclear transfer (NT). Recently introduced zona-free procedures may offer a solution for the latter problem. The most radical approach of these techniques is the so-called handmade cloning (HMC). It does not require micromanipulators because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes, selection of cytoplasts by staining, and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. HMC is a rapid and efficient technique that suits large-scale NT programs. It requires less expertise and time than traditional NT methods and the cost of equipment is significantly less. Production efficiency is high and embryo quality, in terms of pregnancy rates and live births, is not compromised. Although HMC has been developed particularly for bovine NT, the technique is applicable to other species. The method may become a useful tool for both experimental and commercial somatic cell cloning because it allows for standardization of procedures and provides the possibility of automation.

Commercial aspects of cloning and genetic modification in cattle

A range of potential commercial applications of cloning and genetic modification in cattle has been suggested over the last decade. It includes the rapid multiplication of elite genotypes, production of valuable human proteins, altered production characteristics, increased disease resistance and milk with improved nutritional value and processing capabilities. However, an economic return from the sale of product is far from reality in any of these areas. One impediment to achieving economic sustainability is the extremely low efficiency in producing healthy offspring from transferred cloned embryos. Other significant impediments are societal concerns surrounding such technologies, animal welfare issues and regulatory requirements. This review will focus on current biological limitations and technical capabilities in commercial settings, the changes required to allow the production and sale of products at economically sustainable levels, cryopreservation and the progress towards automation of cloning techniques.

Determining the immune mechanisms of protection from AIDS: correlates of immunity and the development of syngeneic macaques: Determining immunity from AIDS

Summary: The AIDS pandemic is a global emergency and a preventive vaccine is urgently needed. CD4 and CD8 T‐cell responses appear important in controlling human immunodeficiency virus (HIV)‐1 in humans and simian immunodeficiency virus (SIV) in macaques. The utility of vaccines that induce high levels of SIV‐ or HIV‐specific T cells has recently become clearer. Since T cells recognize virus‐infected cells rather than free virus, T‐cell‐based vaccines only have the capacity to control infections (non‐sterilizing immunity) and to prevent continuing or persisting infection. An HIV/SIV infection of macaques that is partially controlled by vaccine‐induced T‐cell responses permits a critical window of opportunity for the efficient generation and recruitment of additional T‐ and B‐cell immune responses to the incoming viral inoculum. Although CD8‐depletion experiments in macaques have defined the utility of CD8 T responses in control of SIV infections in macaques, direct evidence on the utility of either CD4 or CD8 T‐cell responses in protective immunity to SIV following vaccination is lacking. The availability of genetically identical macaques would allow cell transfer studies and help define with more certainty the role of cellular immune responses in protection from AIDS. The review also focuses on the development of syngeneic macaques by twinning and cloning technologies.