Expression of recombinant flavin-containing monooxygenases in a baculovirus/insect cell system.

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodopterafrugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.

Transfection of primary cultures of rat hepatocytes.

Five different transfection reagents-calcium phosphate, TransFast Transfection Reagent, Superfect Transfection Reagent, Effectene Transfection Reagent, and Tfx-20--were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids. The efficiency of transfection of each reagent was monitored using the dual luciferase reporter gene assay system. Superfect Transfection Reagent, Effectene Transfection Reagent and Tfx-20 were the most effective for the transfection of primary hepatocytes and gave comparable transfection efficiencies. Calcium phosphate was found to be the least effective transfection reagent and gave the most variable transfection results. Tfx-20 gave the least variable transfection results when different hepatocyte preparations were compared.

Determination of cellular localization of expression of flavin-containing monooxygenase genes in mouse tissues by in situ hybridization.

Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from FMO cDNA templates, the preparation of paraffin wax-embedded and cryostat tissue sections, the hybridization of RNA probes to tissue sections, and the specific detection of hybridized probes using an antibody to DIG.

Characterization of targeted mouse embryonic stem cell chromosomes: karyotyping and fluorescence in situ hybridization of metaphase spreads.

The manipulation of genes in mouse embryonic stem (ES) cells can result in chromosome abnormalities. This chapter describes methods for karyotyping of the manipulated ES cell line before injection into blastocysts and the use of fluorescence in situ hybridization to confirm the deletion of a targeted gene. The method is illustrated by describing how an ES cell line targeted for the deletion of Fmo genes was characterized.

Deletion of genes from the mouse genome using Cre/loxP technology.

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmol, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.

Microinjection of targeted embryonic stem cells and establishment of knockout mouse lines for Fmo genes.

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.