GFP Production Research Articles

Single-cell variability in growing Saccharomyces cerevisiae cell populations measured with automated flow cytometry

Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 μm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible “fingerprint” of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability.

Single-cell variability in growing Saccharomyces cerevisiae cell populations measured with automated flow cytometry

Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 μm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible “fingerprint” of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability.

Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells.

Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs). This non-hr ori is located within the coding sequence of the non-essential p94 gene. We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells. Using homologous ET recombination in E. coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid). All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed. This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging. However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production. The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.

Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria.

The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the Pxyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the Pxyn promoter was replaced with the PxylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population. High-level expression of these reporter constructs in L. monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines. The gfp3+ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.

Regulation of Expression of IL-4 Alleles : Analysis Using a Chimeric GFP/IL-4 Gene

CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments.

A bacterial cloning vector using a mutated Aequorea green fluorescent protein as an indicator

The bacterial cloning vector, pGreenscript A, derived from the mutated Aequorea green fluorescent protein (GFP-S65A) gene, when expressed in E. coli produced colonies that showed yellow color under daylight and strong green fluorescence under long-wave ultraviolet light. The vector was used to select for inserted foreign genes based on the loss of the yellow color/green fluorescence of E. coli cells caused by the insertional inactivation of GFP production.