Improving the transfection efficiency of post-mitotic neurons.

The transfection of post-mitotic cells, including primary cortical and hippocampal neurons, has proven for the most part to be inefficient. Methods such as DNA/Ca++ phosphate co-precipitation, electroporation, cationic lipids and micro-injection are often toxic to the cell and rarely give a transfection efficiency exceeding 3% of the surviving culture. Virus transfection methods using modified viruses such as adeno and semliki-forest virus have been shown to be more efficient but the procedures are often time consuming and virus infections may interfere with protein processing. In this study, we evaluated the transfection efficiency of cells from E18 rat embryonic cortical and hippocampal tissues using three cationic lipids: LIPOFECTAMINE, LIPOFECTAMINE Plus, and LIPOFECTAMINE 2000. The method of transfection was by a traditional reporter gene beta-galactosidase (pCMV.SPORT-beta-gal). Results show that out of the three cationic lipids tested, LIPOFECTAMINE 2000 allows for a significantly higher transfection efficiency. Transfection efficiency with LIPOFECTAMINE or LIPOFECT-AMINE Plus was <3%. In contrast, transfection efficiency with LIPOFECT-AMINE 2000 was approximately 20-25% with the cortical neurons and 25-30% with the hippocampal neurons.

Generation of Recombinant Baculovirus DNA in E.coli Using a Baculovirus Shuttle Vector.

Baculovirus vectors are now widely used to direct the expression of heterologous genes in cultured insect cells and insect larvae. In most cases, heterologous genes placed under transcriptional control of the polyhedrm promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) are abundantly expressed during the late stages of infection. The recombinant proteins are usually soluble and functionally similar to their authentic counterparts (l-7). In the following sections, recent advances in the development of baculovtrus vectors, particularly the baculovn-us shuttle vector system, will be described.

The SFV Gene Expression System.

The SFV Expression System is a DNA expression system used to produce recombinant protein in eukaryotic cells (1,2). The SFV system is based on the Semliki Forest Vnus (SFV), which has several features that provide distinct advantages for a good cDNA expression system. These are: 1. The SFV RNA genome has a positive polarity and thus functions directly as an mRNA. Infectious RNA molecules can be therefore obtained by transcription from a full-length cDNA copy of the genome, 2. The SFV RNA molecule codes for its own RNA rephcase. Consequently, within a few hours after infection, up to 200,000 copies of the plus-RNAs are made in a single cell (3), 3 SFV replication occurs in the cell cytoplasm, where the virus replicase transcribes and caps the subgenomes for production of the structural proteins (4) This eliminates problems, such as mRNA splicing, limitations in transcription factors, problems with capping effctency, and mRNA transport, 4. The cytopathic effects in infected cells appear late during infection. Thus, there is an extensive time window of approx 4-24 h after infection during which a very high expression level of the SFV structural proteins is obtained without significant morphologrcal change of host cells, and 5. Under laboratory conditions, SFV infects a broad range of different cultured cells mammalian, avian, reptilian, amphibian, and insect cells.

Identification of enhancer-like elements in human IFN-gamma genomic DNA.

We have previously shown that transfection of a plasmid clone containing full length human IFN-gamma genomic DNA into a murine T-lymphoblastoid line is followed by basal expression of the transfected gene, with increased transcription occurring upon stimulation of the cells with either phorbol ester or IL-2. In addition, upon transfection of this DNA into murine fibroblasts, high level constitutive transcription was observed. In contrast to the results obtained under tissue culture conditions, introduction of the same DNA into the mouse germline resulted in tissue-specific expression of the transgene. We now report identification of a region 500-bp 5' of the human IFN-gamma TATAA box that has strong, PMA-inducible, enhancer-like activity when linked to a reporter gene (CAT) and transfected into a murine T cell line. However, when the same region of IFN-gamma genomic DNA was introduced into NIH-3T3 cells, no enhancer activity was detected either in the presence or absence of PMA. We have further found that an intronic region of the IFN-gamma genomic DNA (nucleotides 405-674) also contains enhancer activity that is functional in either fibroblasts or T cells. Enhancer activity of the intronic region is also PMA-inducible in the mouse T cells but constitutive in fibroblasts. Collectively, our observations suggest that control of human IFN-gamma gene expression is complex, involving noncontiguous regulatory domains in both 5' flanking and intronic regions of that gene.