Magnetofection of human somatic cells with magnetite and cobalt ferrospinel nanoparticles.

Superparamagnetic nanoparticles varying by their chemical composition and synthesis method were used to transfer DNA into somatic cells under the influence of constant magnetic field (method of magnetofection). Magnetite particles obtained by mechanochemical synthesis ensured higher expression of the marker gene GFP (evaluated by fluorescence intensity of the cell lysate) then particles of ferric oxide obtained by chemical co-precipitation and cobalt ferrospinel particles obtained by the mechanochemical method.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases.

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases

Embryonic stem cells derived from morulae, inner cell mass, and blastocysts of mink: comparisons of their pluripotencies.

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the ICM with both Xs active; 70-100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical "cystic" mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation.

Isolation and cultivation of blastocyst-derived stem cell lines from American mink (Mustela vison).

Ten embryonic stem (ES) cell lines from mink blastocysts were isolated and characterized. All the lines had a normal diploid karyotype; of the ten lines studied, five had the XX and five had the XY constitution. Testing of the pluripotency of the ES-like cells demonstrated that 1) among four lines of genotype XX, and X was late-replicating in three; both Xs were active in about one-third of cells of line MES8, and analysis of glucose-6-phosphate dehydrogenase revealed no dosage compensation for the X-linked gene; 2) when cultured in suspension, the majority of lines were capable of forming "simple" embryoid bodies (EB), and two only showed the capacity for forming "cystic" multilayer EBs. However, formation of ectoderm or foci of yolk sac hematopoiesis, a feature of mouse ES cells, was not observed in the "cystic" EB; 3) when cultured as a monolayer without feeder, the ES cells differentiated into either vimentin-positive fibroblast-like cells or cytokeratin-positive epithelial-like cells (less frequently); neural cells appeared in two lines; 4) when injected into athymic mice, only one of the four tested lines gave rise to tumors. These were fibrosarcomas composed of fibroblast-like cells, with an admixture of smooth muscular elements and stray islets of epithelial tissue; (5) when the ES cells of line MES1 were injected into 102 blastocyst cavities and subsequently transplanted into foster mothers, we obtained 30 offspring. Analysis of the biochemical markers and coat color did not demonstrate the presence of chimaeras among offspring. Thus the cell lines derived from mink blastocysts are true ES cells. However, their pluripotential capacities are restricted.

Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP, and PGP in mink: comparison with man and mouse.

Segregation of mink biochemical markers uridine 5'-monophosphate phosphohydrolase-2 (UMPH2), adenine phosphoribosyltransferase (APRT), phosphoserine phosphatase (PSP), phosphoglycolate phosphatase (PGP), peptidases D (PEPD) and S (PEPS), as well as mink chromosomes, was investigated in a set of mink x mouse hybrid clones. The results obtained allowed us to make the following mink gene assignments: UMPH2, chromosome 8; PEPD and APRT, chromosome 7; PEPS, chromosome 6; and PSP and PGP, chromosome 14. The latter two genes are the first known markers for mink chromosome 14. For regional mapping, UMPH2 was analyzed in mouse cell clones transformed by means of mink metaphase chromosomes (Gradov et al., 1985) and also in mink x mouse hybrid clones carrying fragments of mink chromosome 8 of different sizes. Based on the data obtained, the gene for UMPH2 was assigned to the region 8pter----p26 of mink chromosome 8. The present data is compared with that previously established for man and mouse with reference to the conservation of syntenic gene groups and G-band homoeologies of chromosomes in mammals.

Transfer of mink genes into mouse cells by means of isolated lipid-encapsulated nuclei.

A method for gene transfer by means of interphase nuclei encapsulated within lipid membranes was developed. The method was based on passage of interphase nuclei through a layer of organic solvents containing phospholipids. Evidence was obtained indicating that the nuclei become surrounded by a protective phospholipid membrane: measurements of bound labelled or non-labelled phospholipids; decrease in the permeability of lipid-encapsulated nuclei for high molecular compounds; visualization by direct electron microscopy. Lipid-encapsulated nuclei of mink fibroblasts were used for transformation of mutant mouse LMTK- cells (deficient for thymidine kinase). The frequency of occurrence of HAT-resistant colonies/recipient cell was 1.9 X 10(-5). Biochemical analysis of 14 independent clones demonstrated that they all contained TK1 of mink origin. Analysis of 15 other biochemical markers located on 12 of the mink chromosomes revealed the activities of mink galactokinase (a syntenic marker) in 5 transformed clones, and that of mink aconitase-1 (the marker of mink chromosome 12) in 1 clone. No cytogenetically visible donor chromosomes were identified in the transformed clones. Nine transformed clones were tested for the stability of the TK+ phenotype; of these, the phenotype was expressed stably in 3 and unstably in 6. The method suggested is similar to the gene transfer procedure using total DNA. Its advantage is in ensuring efficient gene transfer and donor DNA integrity.

Regional assignment of the genes for TK1, GALK, ALDC, and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer.

A panel of clones of mink-Chinese hamster somatic cell hybrids was analysed to obtain data for assigning the genes for thymidine kinase-1 (TK1), galactokinase (GALK), subunit C of aldolase (ALDC), and esterase D (ESD) to specific mink chromosomes. The results demonstrate that the genes for TK1, GALK, ALDC and ESD are syntenic and located on mink chromosome 8. Prometaphase analysis of transformed mouse cells obtained by transfer of mink genes by means of metaphase chromosomes demonstrated the presence of mink chromosome 8 fragments of different sizes in some of the independent transformants. Segregation analysis of these fragments and mink TK1, GALK, ALDC and ESD allowed us to assign the genes for TK1 and GALK to 8p24, ALDC to pter-8p25, and ESD to 8q24-8qter.

Cotransfer and phenotypic stabilisation of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer.

By means of metaphase chromosomes, the genes for mink thymidine kinase (TK) and hypoxanthine-phosphoribosyltransferase (HPRT) were transferred to mutant mouse cells, LMTK-, A9 (HPRT-) and teratocarcinoma cells, PCC4-aza 1 (HPRT-). Eighteen colonies were isolated from LMTK- (series A), 9 from A9 (series B) and none from PCC4-aza 1. The transformed clones contained mink TK or HPRT. Analysis of syntenic markers in series B demonstrated that one clone contained mink glucose-6-phosphate dehydrogenase (G6PD) and the other alpha-galactosidase; in series A, nine clones contained mink galactokinase (GALK) and six mink aldolase C (ALDC). Analysis of 12 asyntenic markers located in ten mink chromosomes showed the presence of only aconitase-1 (ACON1) (the marker of mink chromosome 12) in three clones of series A. The clones lost mink ACON1 between the fifth to tenth passages. Cytogenetic analysis established the presence of a fragment of mink chromosome 8 in eight clones of series A, but not in series B. The clones of series A lost mink TK together with mink GALK and ALDC during back-selection; in B, back-selection retained mink G6PD. No stable TK+ phenotype was detected in clones with a visible fragment of mink chromosome 8. Stability analysis demonstrated that about half of the clones of series B have stable HPRT+ phenotype whereas only three clones of series A have stable TK+ phenotype. It is suggested that the recipient cells, LMTK- and A9, differ in their competence for genetic transformation and integration of foreign genes.