Bicistronic expression of ecdysone-inducible receptors in mammalian cells.

The recent emergence of inducible expression systems for mammalian cells has greatly facilitated the in vivo analysis of gene function. The ecdysone-inducible expression system is particularly attractive because of (i) extremely low basal expression and high-level induced expression, (ii) the lack of pleiotropic effects caused by the inducer or activator, and (iii) the rapid penetrance and clearance of the inducer. Here, we describe an improved receptor expression vector. The required ecdysone receptor proteins (VgEcR and RXR) are co-expressed from a bicistronic cytomegalovirus (CMV) expression cassette in the vector pERV3. The CMV promoter in this vector can be readily replaced with a cell type-specific promoter of interest. Using the ecdysone analogs, muristerone A or ponasterone A, induction ratios of up to three orders of magnitude were attained in the transient transfection assays and in a cell line stably transformed with both pERV3 and an ecdysone-inducible reporter vector. Fine control of luciferase expression was achieved bv varying both the induction time and inducer concentration. Here, we describe a set of cell lines stably transformed with the vector pERV3, in which the ecdysone receptors are expressed at optimal levels for the high-level induction of gene expression.

An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions.

We describe a T7-based Escherichia coli expression vector in which protein coding sequence is seamlessly fused to the N-terminal calmodulin-binding peptide (CBP) purification tag. We combined the use of the site-specific protease enterokinase (EK) and the type IIs restriction enzyme Eam1104 I, which cleave outside their respective (amino acid and nucleotide) target sequences, such that any amino acid sequence may be fused directly C-terminal to the EK cleavage site without codon constraints conferred by the cloning method. PCR products are cloned using ligation-dependent or ligation-independent methods with high cloning efficiencies (>10(6) cfu/microg vector), allowing production of insert quantities sufficient for several cloning experiments with a limited number of PCR cycles, resulting in a significant time-savings and reduced likelihood of accumulating PCR-derived mutations. CBP fusion proteins are expressed to high levels when the CBP peptide is positioned at the N-terminus. CBP binds to calmodulin with nanomolar affinity, and fusion proteins are purified to near homogeneity from crude extracts with one pass through calmodulin affinity resin using gentle binding and elution conditions. We show high efficiency seamless cloning of three inserts into the pCAL-n-EK vector, including one encoding the protein c-Jun N-terminal kinase (JNK). CBP-EK-JNK fusion protein was synthesized to 10-20 mg/liter culture and purified to near homogeneity in one step with calmodulin affinity resin. The fusion tag was efficiently removed with EK to yield active JNK with native N-terminal amino acid sequence.

Parameters affecting the use of the lac repressor system in eukaryotic cells and transgenic animals.

Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.

Development of a rat cell line containing stably integrated copies of a lambda/lacI shuttle vector.

A rat embryo cultured cell line was generated that carries stably integrated copies of a lambda/lacI shuttle vector, containing the lacI gene as a mutational target. After the desired treatment of the cells, this vector can be rapidly and efficiently recovered from the cell DNA by in vitro packaging and then screened for mutations in the lacI gene, using bacterial detection systems. The vector is identical to that integrated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis. Characterization of the cell line by fluorescence in situ hybridization showed that the phage DNA is integrated at two distinct sites on separate chromosomes at approximately 50-70 copies per cell and the cell line is polyploid. The rescue efficiency is approximately 100,000 pfu/micrograms of genomic DNA. To examine the ability of the cell line to detect mutations in the lacI gene, the cells were treated with 100 micrograms/ml of the direct-acting alkylating agent N-methyl-N-nitrosourea (MNU) for 30 min at 37 degrees C and grown to confluence. The shuttle vector was rescued from untreated and mutagen treated cells, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10(-5) and 92.7 x 10(-5), respectively. The cell line can be used to detect mutations in the lacI gene, followed by recovery of mutants for sequence analysis. The cell line may be valuable for short-term in vitro mutagenesis studies, oncogene and tumor suppressor studies, and DNA repair studies.

Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation.

Eukaryotic expression vectors designed to produce E. coli Lac repressor protein targeted to the nucleus of mammalian cells were constructed. These constructions carry the lac repressor gene (lacI) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a. When the NLS's were fused to the lacI gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified LacI protein. Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity. When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the LacI protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected. The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacI behind the nine base pair linker, AGC AGC CTG (ser-ser-leu). This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction. The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-terminus of the protein. These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein. This modified lacI gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells.

Analysis of inducers of the E.coli lac repressor system in mammalian cells and whole animals.

Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system. These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers. In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours. Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration. Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG. In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes. HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor. These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.