Transgenic markers for mammalian chimeras

Summary We describe the construction of aggregation chimeras between normal and transgenic embryos containing multiple copies of mouse -globin genes. The transgenic component of the chimeras is then detected in tissue sections by a DNA-DNA in situ hybridization technique, using a biotinylated DNA -globin probe and an avidin-linked alkaline phosphatase detection system. The general advantages of transgenic markers for chimeras are discussed.     

Untersuchung der eiweißspindeln des kakteen-X-Virus mit fluoreszierenden Antikörpern

A?koliv na zá kladě mnoha pokus? se p? edpokládalo, ?e tzv. bÍlkovinná v?etena v buňkách tzn. buně?né inkluse X-viru kaktus? (Ca XV), jsou slo?ena z ?etních prodlou?ených ?ásti Ca XV, p?esto to dosud nebylo proká zá no. Proto jsme se pokusili pomocÍ fluoreskujÍcÍch protilátek doká ?at, ?e bilkovinná v?etena jsou skute?ně agregáty virových ?ástic. V těto práci jsme pouzili tzv. nep?Ímé metody. Nejprve jsme p? sobili na buňky obsahujÍci tato v?etena homologiokým antisé rem proti Ca XV, zÍskanym imunizacÍ králÍk? a teprve potom jsme buňky vlo?ili do roztoku fluoreskujicÍch protilátek proti králicimu γglobulinu. BÍlkovinná v?etena svitila potom ve fluorescen?nÍm mikroskopu silně ?lutozeleně (bylopou?ito fluoresceinisothiocyaná tu). Tato fluorescence ná m uká zala, ?e nastala pozitivnÍ reakce a ?e bÍlkovianá v?etena jsou slo?ena z virových ?ástic. ?etné kontrolnÍ pokusy potvrdily ná? základnÍ pokus.     

Completion of mouse embryogenesis requires both the maternal and paternal genomes

Transplantation of pronuclei between one-cell-stage embryos was used to construct diploid mouse embryos with two female pronuclei ( biparental gynogenones ) or two male pronuclei ( biparental androgenones ). The ability of these embryos to develop to term was compared with control nuclear-transplant embryos in which the male or the female pronucleus was replaced with an isoparental pronucleus from another embryo. The results show that diploid biparental gynogenetic and androgenetic embryos do not complete normal embryogenesis, whereas control nuclear transplant embryos do. We conclude that the maternal and paternal contributions to the embryonic genome in mammals are not equivalent and that a diploid genome derived from only one of the two parental sexes is incapable of supporting complete embryogenesis.     

The beneficial effect EDTA on development of mouse one-cell embryos in chemically defined medium.

An improved methology for culturing noninbred (ICR) mouse one-cell embryos is described. The successful development of one-cell embryos into blastocysts in chemically defined (Whitten's) medium was significantly enhanced by the presence of EDTA. More than 70% of ICR one-cell embryos developed into blastocysts in Whitten's medium in the presence of 10.8 μM EDTA, while, without EDTA, only 15–30% of embryos reached blastocyst stage. A concentration of 10.8 μM EDTA also promoted the development of 65–90% of inbred $\text{C57BL}\text{6}$ one-cell embryos in Whitten's medium. This beneficial role of EDTA is probably related to the chelation of some metal ion(s) other than Ca²⁺ or Mg²⁺.     

Identification of noncollagenous basement membrane glycopolypeptides synthesized by mouse parietal entoderm and an entodermal cell line

Using two-dimensional gel electrophoresis, we have identified two noncollagenous basement membrane (BM) glycopolypeptides which are synthesized by the mouse teratocarcinoma-derived parietal yolk sac (PYS) cell line. These glycopolypeptides have molecular weights of about 200,000 and isoelectric points of about 5.6. Polypeptides with identical parameters are synthesized by the parietal entodermal cells of mouse embryos and are found in Reichert's membrane. Pluripotent embryonal carcinoma cells (ECC) synthesize considerable amounts of the two polypeptides, whereas the yield from nullipotent ECC is negligible. The treatment of nullipotent F9 cells with retinoic acid, which induces entodermal differentiation, activates the synthesis of these polypeptides. These results indicate that the two polypeptides can be used as markers of parietal entoderm differentiation.     

The effect of phosphatidylcholine depletion on biochemical and physical properties of a Neurospora crassa membrane mutant

By using the choline starvation process it is possible to deplete the membranes of Neurospora crassa choline auxotroph $\text{chol-1}$ of phosphatidylcholine, without affecting the viability of germinated spores or whole mycelium. Spin label probes were used to examine the possible dependence of the physical state of cellular lipids on the presence of phosphatidylcholine in the membranes.Increased freedom of rotational motion of lipid soluble probes was regularly detected in choline-starved mycelium. The accumulation of neutral lipids (mostly triglycerides) in bulk form was also observed during the choline starvation process. The experiments with isolated and separated lipid classes indicated that the observed increase in fluidity of lipids in choline-starved mycelium is partly due to the difference in physical properties between bulk lipids and membrane lipids. Spin label probe 2N4 ($\text{2-}\text{propyl}\text{-2,5,5-}\text{trimethyl-oxazolidine}\text{-N-}\text{oxyl}$), which can partition at the membrane-water interface, exhibited easier partitioning among membrane lipids of choline-starved mycelium.     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.