Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.

Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8-9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 10(8) cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats.

The cloning of the human follicle stimulating hormone receptor and its expression in COS-7, CHO, and Y-1 cells.

Follicle stimulating hormone (FSH) receptor clones were isolated from a human testis cDNA library. Characterization of the cDNA clones showed that the DNA and predicted amino acid sequences of the long open reading frame differed from a previously published human ovarian FSH receptor sequence (Minegish et al. (1991) Biochem. Biophys. Res. Commun. 175, 1125-1130) by seven nucleotides and five amino acids. A human FSH receptor splice variant was also identified and characterized. A full-length human FSH receptor cDNA was engineered for expression in COS-7, CHO, and Y-1 cells. In transient transfections of COS-7 cells and stable transfections of Y-1 cells, efficient FSH receptor mRNA accumulation and isolation of FSH-responsive cell lines occurred only when an intron was included in the 5' untranslated region of the FSH receptor transcription unit. Y-1 cells stably transfected with the FSH receptor responded to FSH treatment by rounding up and by synthesizing increased amounts of progesterone. Stably transfected CHO cell lines, which responded to FSH by synthesizing increased amounts of cAMP, were isolated irrespective of the presence of the heterologous intron. The FSH-responsive CHO and Y-1 cell lines may be suitable for the development of better in vitro FSH bioassays. These cells also constitute a convenient source of human FSH receptor protein for use in radioreceptor assays and in studies of receptor-ligand interactions.

Antarctic fish tubulins: heterogeneity, structure, amino acid compositions and charge.

1. Tubulins purified from the brain tissues of three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus) contain equimolar quantities of the alpha and beta chains and are free of microtubule-associated proteins (MAPs) and other non-tubulin proteins. 2. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into 15-20 distinct variants. The range of isoelectric points displayed by the Antarctic fish tubulins (5.30-5.75) is slightly more basic than that of bovine brain tubulin (5.25-5.60). 3. Peptide mapping demonstrated that tubulins from the Antarctic fishes and the cow differ in structure. 4. The amino acid compositions of piscine and mammalian tubulins are similar, but the Antarctic fish tubulins apparently contain fewer glutamyl and/or glutaminyl residues than do tubulins from the temperate channel catfish (Ictalurus punctatus) and the cow. 5. Native tubulin from N. coriiceps neglecta possesses 1-2 fewer net negative charges per tubulin dimer than does bovine tubulin. 6. We suggest that the enhanced assembly of Antarctic fish tubulins at low temperatures (-2 to +2 degrees C) results from adaptive, perhaps subtle, changes in their tubulin subunits.

Heterogeneity and structure of brain tubulins from cold-adapted Antarctic fishes. Comparison to brain tubulins from a temperate fish and a mammal.

Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.