The aim of this study was to compare the efficiency of goat cloning by using cell lineages from distinct transgenic backgrounds. Primary fibroblast skin cell cultures from 2 females (allogeneic), transgenic for the human lysozyme gene (hLZ), were established following standard procedures. Cells from one hLZ genotype were used for the establishment of 2 double transgenic syngeneic cell lines by cell transfection (Nucleofector®, Lonza, Germany) with transgene cassettes containing either the human glucocerebrosidase gene (hGC) and neomycin resistance gene, or the human lactoferrin gene (hLF) with no selection gene. The hGC-transfected hLZ cells were antibiotic-selected (G418, Sigma-Aldrich, St. Louis, MO, USA) until the isolation of positive cell colonies, whereas hLF-transfected hLZ cells were seeded onto 100-mm culture plates (100 cells/plate) to allow colony outgrowth from individual cells. Isolated colonies were screened by PCR using specific primers for each transgene (hGC or hLF) and for hLZ and GAPDH (controls). Positive cells from one hLZ-hGC and one hLZ-hLF colony were used for cloning at passage 9, whereas hLZ cells from the other genotype were at passage 4. Cells were synchronized by high confluence and 24 h of serum starvation. Goat cloning was performed according to standard procedures (Feltrin et al. 2012 Reprod. Fertil. Dev. 25, 163). Briefly, cumulus-oocyte complexes from abattoir ovaries were in vitro-matured for 20 h. Oocyte enucleation and hLZ, hLZ-hGC, or hLZ-hLF donor cell insertion were done by micromanipulation. Reconstructed structures were fused by two 1.2-KV cm–1 DC pulses for 20 μs. Cloned embryos were cultured for 1 h in cytochalasin B and then activated in ionomycin/6-DMAP. After 12 h of in vitro culture in G-1™ medium (Vitrolife, USA), 1-cell stage embryos were transferred into the oviduct of synchronous females (Keefer et al. 2002 Biol. Reprod. 66, 199-203). Pregnancy diagnosis was performed by ultrasonography on Day 30, with weekly monitoring afterwards. Preliminary data from 6 replicates were analysed by the chi-square test (P < 0.05). Maturation rate and survival after enucleation were 42.8% (610/1425) and 72.9% (291/399), respectively. A total of 271 structures were reconstructed using the 3 donor cell lines. Fusion rates did not differ between hLZ (59.5%), hLZ-hGC (47.5%), and hLZ-hLF (48.5%) groups. A total of 68 hLZ, 92 hLZ-hGC, and 39 hLZ-hLF-derived embryos were transferred to 5, 7, and 3 recipients, respectively. No pregnancies were detected with the use of hLZ and hLZ-hLF cells. However, 3 pregnancies (one nonviable) were detected on Day 30 with hLZ-hGC cells (42.9%), with both viable pregnancies lost on Days 40 and 130 of gestation. Molecular analyses confirmed both concepti as transgenic clones from the hLZ-hGC cell line. In summary, antibiotic selection of positive colonies was effective at maintaining cell viability, with a positive response when used for cloning. Replications are in progress to evaluate the effect of cell colony isolation from individual cells (e.g. hLZ-hLF cells) on cell viability over time and on cloning outcome.
Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.