Nucleoplasmin facilitates reprogramming and in vivo development of bovine nuclear transfer embryos.

Successful cloning by somatic cell nuclear transfer (NT) involves an oocyte-driven transition in gene expression from an inherited somatic pattern, to an embryonic form, during early development. This reprogramming of gene expression is thought to require the remodeling of somatic chromatin and as such, faulty and/or incomplete chromatin remodeling may contribute to the aberrant gene expression and abnormal development observed in NT embryos. We used a novel approach to supplement the oocyte with chromatin remodeling factors and determined the impact of these molecules on gene expression and development of bovine NT embryos. Nucleoplasmin (NPL) or polyglutamic acid (PGA) was injected into bovine oocytes at different concentrations, either before (pre-NT) or after (post-NT) NT. Pre-implantation embryos were then transferred to bovine recipients to assess in vivo development. Microinjection of remodeling factors resulted in apparent differences in the rate of blastocyst development and in pregnancy initiation rates in both NPL- and PGA-injected embryos, and these differences were dependent on factor concentration and/or the time of injection. Post-NT NPL-injected embryos that produced the highest rate of pregnancy also demonstrated differentially expressed genes relative to pre-NT NPL embryos and control NT embryos, both of which had lower pregnancy rates. Over 200 genes were upregulated following post-NT NPL injection. Several of these genes were previously shown to be downregulated in NT embryos when compared to bovine IVF embryos. These data suggest that addition of chromatin remodeling factors to the oocyte may improve development of NT embryos by facilitating reprogramming of the somatic nucleus.

Identification of differentially expressed genes in individual bovine preimplantation embryos produced by nuclear transfer: improper reprogramming of genes required for development.

Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and alpha-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.

Production of cloned cattle from in vitro systems.

The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.

Ontogeny of cloned cattle to lactation.

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.