Characterisation of eGFP-transgenic BALB/c mouse strain established by lentiviral transgenesis

Lentiviral technology is a powerful tool for the creation of stable transgenic animals. However, uncertainties have remained whether constitutive promoters resist long-term silencing. We used concentrated HIV-1 based lentiviral vectors to create stable transgenic BALB/c mice by perivitelline injection. In our vectors eGFP expression was driven by the human EF1α promoter. The established transgenic animals were analyzed for eGFP expression by in vivo fluorescence imaging, PCR, histology and flow-cytometry. eGFP expression showed even distribution without mosaicism; however, tissue-dependent differences of eGFP expression were observed. Up to the sixth generation only one newborn showed eGFP inactivation. eGFP + transgenic bone marrow cells efficiently provided long-term haemopoietic repopulation in radiation chimeras, regenerating all bone marrow-derived lineages with eGFP + cells with distinct eGFP expression profiles. The established eGFP + BALB/c mouse strain is expected to be extremely useful in various immunological experiments.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

An unusual radiation-induced G2 arrest in the zygote of the BALB/c mouse strain

Female mice of the BALB/c strain were superovulated, mated with males of the same strain, and irradiated with 1 Gy of X-rays at hourly intervals during the first cell cycle of the embryos. Two types of effects were found in the embryos, depending on the time of X-irradiation. When irradiation was delivered between 14 and 21 h after human chorionic gonadotrophin (hCG) injection, cultured two-cell embryos developed normally up to the morula stage, where a high mortality occurred. On the other hand, when irradiation was given between 17 and 24 h after hCG injection, a high proportion of the eggs was unable to cleave and remained blocked at the one-cell stage. Cytofluorometric analysis of the pronuclear DNA content of uncleaved zygotes showed that DNA synthesis was unaffected by X-irradiation, and that they were blocked in G2 phase of the first cell cycle. Similar studies on other strains, as well as reciprocal crosses between BALB/c and F1(female BALB/c X male C57 BLACK) mice showed that the 'one-cell block' is determined by the maternal genotype and results most probably from a direct action of X-rays on a radiosensitive cytoplasmic factor necessary for the first embryonic cell division, and appearing 17 h after hCG injection. A high proportion of blocked zygotes (30-40 per cent) recovered partially, cleaved with a delay of about 20 h, and died soon after, almost none of them being able to reach the blastocyst stage. At the time of maximum radiosensitivity, the LD50 for development up to the blastocyst stage was 0.95 Gy.     

The structure of apolipoprotein A-II in discoidal high density lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.     

Nucleotide sequence of the BALB/c mouse beta-globin complex

The nucleotide sequence of 55,856 base-pairs containing all seven beta-globin homologous structures from chromosome 7 of the BALB/c mouse is reported. This sequence links together previously published sequences of the beta-globin genes, pseudogenes and repetitive elements. Using low stringency computer searches, we found no additional beta-globin homologous sequences, but did find many more long interspersed repetitive sequences (L1) than predicted by hybridization. L1 is a major component of the mouse beta-globin complex with at least 15 elements comprising about 22% of the reported sequence. Most open reading frames greater than 300 base-pairs in the cluster overlap with L1 repeats or globin genes. Polypurine, polypyrimidine and alternating purine/pyrimidine tracts are not evenly dispersed throughout the complex, but they do not appear to be excluded from or restricted to particular regions. Several regions of intergenic homology were detected in dot-plot comparisons of the mouse sequence with itself and with the human beta-globin sequence. The significance of these homologies is unclear, but these regions are candidates for further study in functional assays in erythroid cell lines or transgenic animals.     

Mechanisms mediating insulin resistance in transgenic mice overexpressing mouse apolipoprotein A-II

We previously demonstrated that transgenic mice overexpressing mouse apolipoprotein A-II (apoA-II) exhibit several traits associated with the insulin resistance (IR) syndrome, including increased atherosclerosis, hypertriglyceridemia, obesity, and IR. The skeletal muscle appeared to be the insulin-resistant tissue in the apoA-II transgenic mice. We now demonstrate a decrease in FA oxidation in skeletal muscle of apoA-II transgenic mice, consistent with reports that decreased skeletal muscle FA oxidation is associated with increased skeletal muscle triglyceride accumulation, skeletal muscle IR, and obesity. The decrease in FA oxidation is not due to decreased carnitine palmitoyltransferase 1 activity, because oxidation of palmitate and octanoate were similarly decreased. Quantitative RT-PCR analysis of gene expression demonstrated that the decrease in FA oxidation may be explained by a decrease in medium chain acyl-CoA dehydrogenase. We previously demonstrated that HDLs from apoA-II transgenic mice exhibit reduced binding to CD36, a scavenger receptor involved in FA metabolism. However, studies of combined apoA-II transgenic and CD36 knockout mice suggest that the major effects of apoA-II are independent of CD36. Rosiglitazone treatment significantly ameliorated IR in the apoA-II transgenic mice, suggesting that the underlying mechanisms of IR in this animal model may share common features with certain types of human IR.     

CBXC: a set of recombinant inbred strains between CBA/Ca and BALB/c

A new set of nine recombinant inbred strains designated CBXC-1 to CBXC-9 has been developed from a cross between CBA/Ca female and BALB/c male mice. All of the strains have been brother x sister mated for more than 20 generations, and have been characterized at eight electrophoretic, one immunological and two coat colour loci at which the progenitor strains differ. This set of strains should be useful in investigating the genetics of any characters which differ between the two progenitor strains.     

Development and characterization of the IOR/Hab inbred mouse strain

The IOR/Hab inbred mouse strain, developed and maintained in the National Institute of Oncology and Radiobiology, is the first inbred strain to be developed in Cuba. It has a high fertility in this tropical climate. A number of physiological characteristics such as longevity, haematological characteristics, and growth are reported. Other experiments and results concerning spontaneous tumour frequency suggest that the strain is well adapted to standard environmental conditions, and could be useful for biomedical research.     

Enhancement of cellular immune function during cold adaptation of BALB/c inbred mice.

The cell-mediated immune function of cold-adapted BALB/c inbred mice was studied in experiments of splenic lymphocyte blastogenesis, indicated by tritium-labeled deoxythymidine incorporation and SDS-PAGE autoradiography of synthetic proteins in lymphocytes. Male BALB/c inbred mice were randomly divided into two groups: control (living at 25 degrees C) and cold-exposed (living at 2 degrees C). Results are as follows: in contrast with the control group, there was an obvious fluctuation of cell-mediated immune function in the cold-exposed group at initial cold exposure because of transient stress to cold; then cell-mediated immune function gradually recovered to control level. From Day 15, the cell-mediated immune function of the cold-exposed group was remarkably enhanced. On Day 15, the lymphocyte blastogenesis rate was increased by 20.66% (P less than 0.05), which implies the onset of cold adaptation; on Days 21 and 31, the rates increased by 80.15% (P less than 0.05) and 40.36% (P less than 0.05), respectively. Two to six months later, with continuing cold exposure, the murine lymphocyte blastogenesis rate in the cold-exposed group remained higher than that in the control group. The lymphocyte protein synthesis of the cold-exposed group, indicated by tritium-labeled leucine incorporation, apparently increased on Day 15 and the stimulated rate was 101.47% (P less than 0.05). SDS-PAGE autoradiography of synthetic proteins in lymphocytes demonstrated that after 2 weeks of cold exposure, protein bands were enriched in both quantity and quality. These results are identical to the results obtained from lymphocyte blastogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c.

Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three of four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral DNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded GIX cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype.     

Hydronephrosis in the inbred mouse strain DDD

The inbred mouse strain DDD was found to have an extremely high incidence of hydronephrosis (37/37 in adult males and 12/32 in adult females). The hydronephrosis was classified as open with no definite cause for obstruction. The condition was either unilateral in the right kidney or bilateral. Another feature of the hydronephrotic kidney was circulatory failure. Hydronephrosis in strain DDD mice is considered to be a useful experimental animal model with additional possible use, in investigating disturbances of renal haemodynamics and function.     

Relationship between DNA replicon size and SCE induction in BALB/c and BALB/Mo mouse lymphocytes

We studied the DNA replicon size in BALB/c and BALB/Mo mouse lymphocytes by the method of bromodeoxyuridine photolysis. After treatment of the BALB/Mo lymphocytes in vitro with mitomycin C, the average DNA replicon size appeared to be significantly smaller than that observed in BALB/c lymphocytes treated similarly. In these conditions an increased susceptibility to SCE induction in BALB/Mo lymphocytes had been observed. In the presence of both mitomycin C and cordycepin (an antiviral drug), both the DNA replicon size and the SCE frequency returned to normal values.     

Bacterial colonization of the oral cavity of the BALB/c mouse

The acquisition of the human oral bacterial flora follows a relatively well known sequence of succession that can be influenced by various host factors. These factors have not been studied in the mouse. In the present work, we followed the bacterial colonization of the oral cavity of mice from birth, and tested our mouse model for its suitability in studying the influence of weaning and puberty on the indigenous oral bacterial flora. We observed that the first colonizers were staphylococci, followed by lactobacilli. The proportions of these two predominant bacteria fluctuated for a period of 30–50 days, but evolved towards the proportions previously observed among the indigenous bacterial species of 6–8 week-old BALB/c male mice (predominantly Lactobacillus murinus and Staphylococcus aureus). The weaning period significantly altered the equilibrium among the oral bacterial flora. This equilibrium was not significantly modified during puberty.Offprint requests to: M.C. Lavoie     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

Effects of a bacteriocin from Mycobacterium smegmatis on BALB/3T3 and simian virus 40-transformed BALB/c mouse cells

The effects of a bacteriocin from Mycobacterium smegmatis ATCC 14468 on simian virus 40-transformed BALB/c mouse cells (mKS-A TU-7 cells) and nontransformed BALB/3T3 cells originating from the BALB/c mouse strain were studied. The percentage of nigrosin-unstained (viable) cells in the bacteriocin-treated mKS-A TU-7 cells decreased time-dependently with an increase in the bacteriocin activity. There was a time-dependent decrease in the bacteriocin activity after treatment with the cell membrane preparation from mKS-A TU-7 cells. There was no apparent effect of the bacteriocin on the viability of nontransformed BALB/3T3 cells. Wheat germ agglutinin blocked the toxic effect of bacteriocin on mKS-A TU-7 cells. These results indicate that the higher sensitivity and binding capacity of the tumor cells to the bacteriocin is probably due to the presence of a large amount of N-acetyl-glucosamine or closely related sugar residues with a high affinity for bacteriocin, as compared with normal cells. The bacteriocin produced morphological alterations and inhibition of synthesis of ribonucleic acid, deoxyribonucleic acid and protein in the transformed but not in the nontransformed cells.     

The effect of age on lung structure in male BALB/cNNia inbred mice

Morphometric examination using light, scanning electron, and transmission electron microscopy was performed on the lungs from 32 inbred male BALB/ cNNia mice between 38 days and 28 months of age. Between 38 days and 9 months of age the changes were primarily those of growth. Alveolar multiplication and total elastic-fiber length appeared complete by 38 days of age. The major increase in the number of interalveolar pores occurred by 68 days, but there was a significant further increase from 68 days to 9 months of age. At 9 months, approximately 10% of the alveolar wall was formed by pores. Alveolar ducts, the cylindrical core of air central to alveolar mouths, increased more in diameter than length. Between 9 and 28 months the changes were attributed to aging and were different from those reported in humans and other species. Lung volume, alveolar surface area, and total volume of alveolar wall increased with age; there was no change in mean linear intercept and volume proportion of alveolar and alveolar duct air. Total area of pores increased with age, but their number and area fraction of the alveolar wall did not change. No transmission electron microscopic changes were seen in the alveolar walls. We speculate that the morphometric differences between our animals and those studied in other reports may reflect the fact that our animals were specific-pathogen-free animals and kept under protected circumstances.     

The determination of the oxidation phenotype in inbred C57Bl/6 and BALB/c mice

Antipyrine oxidation was studied in C57BL/6 and BALB/c inbred mice. It was found that C57BL/6 are weak oxidant but BALB/c are strong oxidants of antipyrine. Animals F1 hybrids inherited the high capacity of antipyrine oxidation.