The important steps involved in somatic cell nuclear cloning are shown schematically. Progress in each of the boxed methodologies will be discussed in detail in subsequent sections [33-35]. Italicized labels indicate descriptive markings, not methodological steps per se.

General strategy for generating gene-targeted animal models using somatic cell nuclear cloning. Two major steps include: 1) Targeting gene mutations in appropriate somatic cell nuclear donor and 2) embryo reconstruction and cloning of animal.

Implantation and fetal development of NT-reconstructed ferret embryos. (A and B) Ovaries, oviducts and the uteri from two recipient albino ferrets were dissected at 21 days following transfer of ~25 NT-reconstructed embryos. The swollen regions of the uterus (denoted by arrows) are regions of implanted NT-reconstructed embryos. Recipient albino Jills were mated with vasectomized (sterile) male albino studs. Vasectomized studs were proven sterile by sperm count and mating. Albino oocytes were reconstructed with sable × cinnamon F1 fetal ferret fibroblasts. (C) Fetuses marked 1 and 2 were dissected from the swollen regions of the uterus in Panel B. (D) Normal 3-week ferret fetus developed from in vivo fertilization by normal mating. Embryo #1 appears to have normal 21-day development and is approximately 1.5 cm on the longest axis. Scale marker: Panels A and B, 1.5 cm; Panels C and D, 0.5 cm. Reproduced with permission from a published manuscript by Li et al [35].

Chimeraplast targeting of the G551D mutation to the human CFTR gene in Hela cells. (A) The 25 bp segment homologous to CFTR is bolded, with the mutated nucleotide (G → A) indicated by asterisks. This structure is based on the traditional design with a 5 bp DNA (capitalized and underlined) core region flanked by two 10-nt 2'-O-methyl RNA stretches (lowercased). Only one base-pair change exists in parentheses for generating such an oligo for ferret CFTR. (B, C) Cell lysates prepared with 44 targeted Hela cell clones after G551D chimeric oligonucleotide transfection were amplified by 2 rounds of PCR and analyzed by ASO hybridization against (B) wild-type CFTR and (C) G551D mutant oligonucleotide probes. Three of these cell clones (2b, 3c, and 6a) were highly positive for both the wild-type and the G551D genotype, as indicated by the asterisks. Positive plasmid cDNA controls for the wild-type (1e) and the G551D (1f) CFTR sequences were also run as standards. PCR blanks are shown in 1 g and 1 h. All other wells contain experimental Hela cell clone PCR material. Wells that showed no hybridization to either probe probably contained no DNA material, which was likely due to insufficient cells for efficient PCR.