A short-term culture method for the preparation of chromosome spreads from Rana pipiens.

Chromosomes suitable for karyotyping were produced from short-term cultures of cells from embryonic northern leopard frogs, Rana pipiens, at various growth stages. Embryos were minced in a trypsin solution. The tissue fragments were subsequently cultured in an electrolytic solution containing fetal calf serum and Colcemid. We found this technique to be highly reproducible. In contrast to the conventional squash technique we were able to obtain a greater number of metaphase spreads suitable for identification and analysis of individual chromosomal aberrations.     

Stability of X chromosome differentiation in mouse embryos

Summary By means of a double labeling method with H³-thymidine and 5-bromodeoxyuridine, it was found that the X chromosome showed no sign of change from an allocyclic to an isocyclic state, or vice versa in 6.5- and 7.5-day mouse embryos. Thus, reversal of allocycly may not account for the predominance of cells with the paternally derived X chromosome inactive in the yolk sac and the chorion of the mouse embryo.     

Developmental abnormalities in mouse embryos tetrasomic for chromosome 11: apparent similarity to embryos functionally disomic for the x chromosome

Adopting a mating system involving two different Robertsonian translocations with monobrachial homology, we studied the early development of mouse embryos trisomic or tetrasomic for chromosome 11. A developmental delay of 12-24 hours was evident in trisomic embryos at embryonic day (E)7.5, whereas tetrasomic embryos apparently had stopped growth by E6.5 without formation of extraembryonic structures. This extremely severe developmental abnormality found in tetrasomic embryos is similar to that reported in embryos having two active X chromosomes in extraembryonic cell lineages. Autosomal tetrasomy, but not autosomal trisomy, can lead to such early developmental errors. Thus, a reasonable inference would be that the X chromosome is twice as active as the autosome. Probably, the X chromosome became upregulated in response to the evolutionary necessity of minimizing haplo-insufficiency brought about by miniaturization of the Y chromosome.     

Mutagenicity of isoniazid: Testing for somatic chromosome aberrations in mouse embryos

Summary Isonicotinic acid hydrazide (INH) were given by peroral intubation to pregnant mice of strain C57BL/6Ffm on day 9 of pregnancy, INH was given in the following doses: 0, 5, 25, and 125 mg/kg solved in physiological saline. Cytogenetic analysis of homogenized embryos 6, 12, 24, and 48 h, resp., after treatment of the females did not show any increase of the rate of gaps or chromosomal aberrations.     

A new method for fish chromosome preparation

A new method for the preparation of fish chromosomes from abdominal cavity fluid has been developed. Cells were collected from fish abdominal cavity fluid after an in vivo PHA treatment, and cultured for a short time in medium with colchicine. After 30 min hypotonic treatment for marine fish and 35 min for freshwater fish, slides were prepared by the conventional air-drying method. The advantages ofthe method are: (1) it is technically simple; (2) it produces a reasonably high mitotic index; (3) chromosome spreading is good (4) there is very little cell breakage. Using this method, the chromosomes ofrainbow trout (2n=62); cod (2n=4546) and plaice (2n=46,47 and 48) were investigated.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.     

A chromosome marker for the early detection of mouse embryos carrying the neural tube defect mutation splotch

A major problem in the study of neural tube defects caused by the splotch (Sp) gene in the mouse has been the identification of gene carriers or potentially affected embryos at an early stage of development, since the gene's effects become visible only late in gestation or after birth. To aid in the identification of Sp carriers, we have developed a technique using a Robertsonian translocation as a marker for this gene. The accuracy of identification is reduced by crossing-over between the Sp locus and the centromere but, because of crossover suppression in the particular cross used, there was only 23.2% recombination compared with the known map distance of 36%. Paternal age had no effect on the frequency of recombination, but individual males differed significantly in the degree of crossover suppression.     

A trial of histological preparation of early mouse embryos with the Technovit 7100 system

A reproducible procedure was devised for making histological preparations of early mouse embryos cultured in vitro. This method was characterized by use of a resinous embedding material, Technovit 7100, and a 96-well U-bottomed microplate, which enabled efficient rinsing and dehydration of fixed embryos without loss. Technovit 7100 polymerized or hardened at room temperature slowly enough to allow sedimentation of embryos to the bottom, which enabled more correct prediction of the place of the embryos at sectioning. The present method will lead to easier microscopic observation of early embryos in embryology.     

Sensitivity of the early mouse embryos to antifolic preparation chloridine]

A study was made of antifolic preparation chloridine (2,4-diamino-5-p-chlorphenyl-6-ethylpyrimidine) on the cleavage of CBA mouse embryos. Chloridine failed to influence the development of early mouse embryos in the maternal organism and considerably inhibited the cleavage of embryos explanted into the chloridine-containing medium. Chloridine sensitivity in the mouse and rat embryos was compared and a conclusion was drawn on the absence of interspecies differences in their reaction to teratogen at the level of the embryonic cells.     

A leukocyte culture and chromosome preparation technique for avian species

Summary A technique for avian leukocyte culture and chromosome analysis is described. The method is simple and allows karyotypic analysis by a variety of chromosome banding methods. It is applicable to a wide variety of species and may be useful in determining the genetic sex of monotypic species in captivity or for population studies of specimens in the wild. This work was supported by Medical Research Council (Canada) Grant MA-4655. B. M. B. is a recipient of a Medical Research Council Fellowship.     

Lymphocyte culture for chromosome preparation

A technique is described using separated peripheral blood lymphocytes from fish which yields cultures giving good chromosome spreads suitable for convential karyotyping and the subsequent application of banding techniques.     

Use of the intact mouse skeletal-muscle preparation for metabolic studies. Evaluation of the model.

1. We examined the isolated mouse skeletal-muscle model in vitro, commonly used by many investigators, for its suitability for metabolic studies. 2. Despite the fact that pH, O2 saturation, osmolality and the release of the enzyme creatine kinase remained stable, histochemical studies showed large cores devoid of glycogen, suggesting that the incubated muscle had lost its viability. 3. This study indicates that caution should be exercised when interpreting the results of studies with intact isolated mouse muscles.     

一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.