Cotransfection Method Research Articles

Generation of hybrid human immunodeficiency virus utilizing the cotransfection method and analysis of cellular tropism

Human immunodeficiency viruses (HIV) isolated from infected individuals show tremendous genetic and biologic diversity. To delineate the genetic determinants underlying specific biologic characteristics, such as rate of replication, cytopathic effects, and ability to infect macrophages and T4 lymphoid cells, generation of hybrid HIV using viruses which exhibit distinct biologic features is essential. To develop methods for generating hybrid HIV, we constructed truncated HIV proviral DNA plasmids. Upon digestion with restriction enzymes, these plasmid DNAs were cotransfected into human rhabdomyosarcoma cells to generate hybrid HIV. The hybrid HIVs derived by this method were infectious upon transmission to both phytohemagglutinin-stimulated peripheral blood lymphocytes and established human leukemic T-cell lines. The virus derived from molecular clone pHXB2 (HIVHTLV-III) productively infected CEMx174 cells. On the other hand, molecular clone pARV (HIVSF2)-derived virus did not show productive infection of CEMx174 cells when used as a cell-free virus. The hybrid HIV containing the 3' end of the genome from pARV and the 5' end of the genome from pHXB2 was effective in infecting CEMx174 cells, but the converse hybrid containing 5' pARV and 3' pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef play a role in the ability of the virus to infect a certain cell type. The intracellular ligation method should be useful in the analysis of related and unrelated HIV-1 isolates with common restriction enzyme cleavage sites.

An adenoviral vector system for functional identification of nuclear receptor ligands.

A recombinant adenovirus system has been designed that confers glucocorticoid responsiveness upon infected cells in culture. Two mutually dependent viruses are required: a trans-activator virus containing the human glucocorticoid receptor transcription unit and a second receptor virus harboring a glucocorticoid response element linked to the firefly luciferase gene. Another reciprocal pair of viruses has been generated; one member expresses the rat thyroid hormone receptor alpha, while the other contains the luciferase gene regulated by a thyroid hormone-responsive DNA element. Corticosteroid- or thyroid hormone-induced transcription can be efficiently and accurately quantitated from cells coinfected with the appropriate complementary virus pair 20 h after infection in 96-well microtiter plates. This coinfection assay offers a convenient way to measure transcriptional activation by nuclear receptors and has certain key advantages over the commonly used cotransfection method. Its sensitivity and precision make it a practical approach to rapidly identify substances extracted from complex biological samples activating candidate "orphan" nuclear receptor molecules.

Inhibition of Marek's Disease Virus DNA Transfection by a Sequence Containing an Alphaherpesvirus Origin of Replication and Flanking Transcriptional Regulatory Elements

A co-transfection method was used to identify regions of the Marek's disease virus (MDV) genome that inhibit plaque formation when introduced along with total DNA from MDV-infected chicken embryo fibroblasts into secondary chicken embryo fibroblasts. Co-transfections involving MDV plasmids containing the BamHI-D or BamHI-H regions of the genome inhibited plaque formation more than 10-fold. The inhibitory region was localized to a 222-base-pair region that contains a sequence homologous to the consensus origin of replication of alphaherpesviruses. The region also contains several potential transcriptional regulatory elements, including two CCAAT boxes, one Sp1 binding site, and an octamer element. The sequence of this region has been reported previously. Transfection inhibition was also observed for the BamHI-I2 region, although the effect was weaker.

Engineering of an Oryctes baculovirus recombinant: insertion of the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus.

A cotransfection method has been developed for the generation of recombinant Oryctes baculoviruses. The permissive coleopteran cell-line DSIR-HA-1179 was transfected with a mixture of Oryctes baculovirus DNA (strain PV505) and a transfer vector. The transfer vector, a pUC8-based plasmid, contained the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus, flanked by Oryctes baculovirus DNA from the HindIII fragment N. Oryctes baculovirus does not form plaques with DSIR-HA-1179 cells. Therefore an endpoint dilution method was used to screen for, recover and purify recombinants. There was no phenotypic character that could be used for detecting recombinants, so a dot blot assay was used to screen infected cultures for the presence of recombinants. A recombinant generated by this method contained the entire polyhedrin gene inserted at 98.03 map units from the designated start of the Oryctes baculovirus physical map. No evidence of transcription or translation of the polyhedrin gene was obtained.

Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding α 3-subunits of rat brain Na +,K +-ATPase

The cDNAs encoding α 3-subunits of rat brain Na +,K +-ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co-transfection method. Stably transformed cells were selected with 300 μg/ml of neomycin (G-418) for 6 weeks. Northern blot analysis using the 3′-non-translated region of the cDNA as a probe revealed that the α 3 mRNA appeared in transfected cells. Na +,K +-ATPase activity of the transfected cells was twice that of wild-type cells. Regarding ouabain sensitivity, the Na +,K +-ATPase showed two K i values for ouabain (8 × 10 −8 and 4.5 × 10 −5 M) in transfected cells while wild-type cells displayed only the higher value. Ouabain sensitivity of Rb + uptake also demonstrated two K i values in the transfected cells (8 × 10 −8 and 4.5 × 10 −5 M) and a K i in wild-type cells of 4 × 10 −5 M. It is concluded that α 3 is a highly ouabain-sensitive catalytic subunit of Na +,K +-ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the α-subunit rather than the β-subunit. This is the first report on the catalytic charateristics of the α 3 isoform of Na +,K +-ATPase.