Cell cycle analysis of foreign gene (beta-galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter

The cell cycle dependency of foreign gene expression in recombinant mouse L cells was investigated. Two different recombinant mouse L cell lines having the glucocorticoid receptor-encoding gene and the lacZ reporter gene were used in this study. The lacZ gene expression was controlled by the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter in both cell lines. In "M4" cells the gr gene was under the control of another MMTV promoter, but in "R2" cells it was under the control of the constitutive Rous sarcoma virus promoter. These normally attachment-grown cells were adapted to suspension culture, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in exponential growth without any addition of the glucocorticoid inducer, dexamethasone. Cell cycle positions in the S phase were estimated on the basis of DNA content per cell, and position in the G1 phase was estimated on the basis of cell size as measured by pulse-width time of flight. The results showed that beta-galactosidase synthesis occurred through all cell cycle phases, but the expression rate in the G1 phase was much lower than that in the S and G2/M phases in both cell lines. On the basis of cell size analysis, beta-galactosidase expression in M4 cells (with autoinducible promoter) was found to be higher than that in R2 cells (with inducible promoter) during the G1 phase. (c) 1996 John Wiley & Sons, Inc.     

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1(Ser) gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1(Ser). Several genes containing UCA codons are normally expressed at 42 degrees C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1(Ser). In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.     

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL)

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.     

Head and neck cancer cells are efficiently infected by Ad5/35 hybrid virus

BACKGROUND: Clinical gene therapy trials using standard Ad5-based vectors have thus far demonstrated limited efficacy, most likely due to low expression levels of adenoviral receptors on tumor cells. We sought to analyze adenoviral receptor expression levels on primary head and neck squamous cell carcinoma (HNSCC) cells and to determine whether adenoviral retargeting to the CD46 receptor via the Ad5/35 system would increase therapeutic potential for HNSCC. METHODS: We used flow cytometric analyses to determine adenoviral receptor expression levels on nine primary HNSCC cells collected from cancer patients. Adenoviruses Ad5.LacZ and Ad5/35.LacZ were used to analyze the differences in viral transduction both in vitro and in a HNSCC tumor mouse model. RESULTS: Flow cytometric analyses demonstrated uniformly high CD46 expression in all cells studied (85-99%). In contrast, coxsackievirus and adenovirus receptor (CAR) expression was substantially lower and highly variable (1.6-62%). Alpha(v) integrin expression was between 39-98%. In situ stainings for beta-galactosidase gene expression demonstrated that Ad5/35.LacZ was clearly more effective than Ad5.LacZ in transducing primary HNSCC cells. Quantification of beta-galactosidase expression revealed up to 65 times higher transgene expression from Ad5/35.LacZ than Ad5.LacZ. In vivo, beta-galactosidase expression was detected in a substantial area after a single intratumoral injection of Ad5/35.LacZ, whereas injection with Ad5.LacZ resulted in gene expression only in a few cells. CONCLUSIONS: Our results demonstrate that the low and variable CAR expression levels limit the therapeutic efficacy of Ad5-based strategies for HNSCC. In contrast, the effective in vivo transduction capacity of Ad5/35 warrants further development of this vector for the treatment of head and neck cancer.     

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.     

Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth. (c) 1993 John Wiley & Sons, Inc.     

In vivo analysis of Frat1 deficiency suggests compensatory activity of Frat3.

The Frat1 gene was first identified as a proto-oncogene involved in progression of mouse T cell lymphomas. More recently, FRAT/GBP (GSK-3beta Binding Protein) family members have been recognized as critical components of the Wnt signal transduction pathway. In an attempt to gain more insight into the function of Frat1, we have generated Frat1-deficient mice in which most of the coding domain was replaced by a promoterless beta-galactosidase reporter gene. While the pattern of LacZ expression in Frat1(lacZ)/+ mice indicated Frat1 to be expressed in various neural and epithelial tissues, homozygous Frat1(lacZ) mice were apparently normal, healthy and fertile. Tissues of homozygous Frat1(lacZ) mice showed expression of a second mouse Frat gene, designated Frat3. The Frat1 and Frat3 proteins are structurally and functionally very similar, since both Frat1 and Frat3 are capable of inducing a secondary axis in Xenopus embryos. The overlapping expression patterns of Frat1 and Frat3 during murine embryogenesis suggest that the apparent dispensability of Frat1 for proper development may be due to the presence of a second mouse gene encoding a functional Frat protein.     

In situ lacZ retrovirus-marked lymphocytes define a B cell microenvironment in the lymph node medulla.

A recombinant nonreplicating retroviral vector bearing the Escherichia coli lacZ indicator gene was used to mark a population of B cells in situ in murine lymphoid tissue. The retrovirus was surgically injected into popliteal lymph nodes during the primary immune response to DNP-CGG when B cell proliferation in the germinal centers was maximal. LacZ+ cells were initially detected in the perivascular medullary interstitium, where they expanded and persisted up to 2 weeks following retrovirus injection. Migrant lacZ+ B cells were detected in the spleen 3-18 weeks following immunization and resided in the red pulp or marginal zones. Two-color flow cytometric analysis using a fluorogenic substrate for beta-galactosidase revealed that lacZ+ cells bear kappa light chains and that at least 50% of these cells bound the hapten, DNP. Based on their location, life span, migratory capacity, antigenic specificity, and surface immunoglobulin density, lacZ+ cells define a distinct nonfollicular B cell population associated with other late developmental stages of B lymphocytes, including memory and plasma cells.     

Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants.

The lacZ gene of Escherichia coli, coding for beta-galactosidase, is a widely used reporter gene for gene expression studies in microbial and animal systems. To demonstrate that it is also a powerful reporter gene in plants, lacZ was fused to 5' regulatory elements of several genes known to be functional in plant cells. By measuring LacZ activities in transgenic plants containing these gene constructs, we showed that the reporter is correctly monitoring the regulatory properties of the well-characterized promoters fused to lacZ. beta-Galactosidase was assayed directly in plant extracts when they contained high levels of LacZ or, when LacZ was expressed at low level, by separating the endogenous and LacZ activities electrophoretically and detecting the enzymes with a fluorogenic substrate. The most outstanding property of the marker is its amenability to histochemical detection. Due to its stability, LacZ can be fixed in the tissue with glutaraldehyde without loss of activity and detected with high resolution by using XGal. We could reveal expression patterns unnoticed earlier for many of the regulatory elements studied. The chlorophyll a/b binding protein gene, expressed at very high levels in green tissues, is also expressed at a low level in the vascular cylinder of the root. The Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and the TR2' gene was shown to be root specific in the intact plant and stimulated by wounding in the leaf tissue. The TR1' gene, fused to nptII, shows similar characteristics suggesting co-regulation of this tightly linked dual promoter.     

Position effects in mice carrying a lacZ transgene in cis with the beta-globin LCR can be explained by a graded model.

We studied transgenic mice carrying the lacZ reporter gene linked to the erythroid-specific beta-globin promoter and beta-globin locus control region (LCR). Previously, we had demonstrated that the total level of expression of beta-galactosidase enzyme, which is the product of the lacZ gene, varies widely between different transgenic mice due to position effects at the sites of transgene integration. Here, using the X-gal based in situ assay for beta-galactosidase activity, we found that the percent erythroid cells that expressed the transgene also varied widely between the mice. Moreover, a kinetic analysis showed that the average beta-galactosidase content per expressing cell varied both between samples of different transgenic descent and between erythroid cells within each sample, demonstrating that the variable expression of this lacZ transgene was being controlled in a graded manner. These results suggest that the beta-globin LCR enhancers function through a graded model, which is described, rather than the binary mechanism that has been proposed previously for other enhancers.     

Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors

Recent studies have demonstrated the usefulness of dendritic cells (DCs) genetically modified by adenovirus vectors (Ad) to immunotherapy, while sufficient gene transduction into DCs is required for high doses of Ad. The RT-PCR analysis revealed that the relative resistance of DCs to Ad-mediated gene transfer is due to the absence of Coxsackie-adenovirus receptor expression, and that DCs expressed adequate alpha(v)-integrins. Therefore, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob can efficiently transduce and express high levels of the LacZ gene into DCs. The gene delivery by fiber-mutant Ad was more efficient than that by conventional Ad in both murine DC lines and normal human DCs (NHDC). Furthermore, NHDC transduced with fiber-mutant Ad and conventional Ad at 8000-vector particles/cell resulted in a 70-fold difference in beta-galactosidase activity. We propose that alpha(v)-integrin-targeted Ad is a very powerful tool with which to implement DC-based vaccination strategies.     

Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions

After skeletal muscle is denervated, fibroblasts near neuromuscular junctions proliferate more than fibroblasts distant from synaptic sites, and they accumulate adhesive molecules such as tenascin (Gatchalian, C. L., M. Schachner, and J. R. Sanes. 1989. J. Cell Biol. 108:1873-1890). This response could reflect signals that arise perisynaptically after denervation, preexisting differences between perisynaptic and extrasynaptic fibroblasts, or both. Here, we describe a line of transgenic mice in which patterns of transgene expression provide direct evidence for differences between perisynaptic and extrasynaptic fibroblasts in normal muscle. Transgenic mice were generated using regulatory elements from a major histocompatibility complex (MHC) class I gene linked to the Escherichia coli beta-galactosidase (lacZ) gene. Expression of lacZ was detected histochemically. In each of eight lines, lacZ was detected in different subsets of cells, none of which included lymphocytes. In contrast, endogenous MHC is expressed in most tissues and at high levels in lymphocytes. Thus, the MHC gene sequences appeared inactive in the transgene, and lacZ expression was apparently controlled by genomic regulatory elements that were specific for the insertion site. In one line, cells close to the neuromuscular junction were lacZ positive in embryonic and young postnatal mice. Electron microscopy identified these cells as fibroblasts and Schwann cells associated with motor nerve terminals, as well as endoneurial fibroblasts, perineurial cells, and Schwann cells in the distal branches of motor nerves. No intramuscular cells greater than 200 microns from synaptic sites were lacZ positive. These results indicate that there are molecular differences between perisynaptic and extrasynaptic fibroblasts even in normal muscle and that diverse perisynaptic cell types share a specific pattern of gene expression.