Recombinant CHO Cell Line Research Articles

Development of serum-free media for a recombinant CHO cell line producing recombinant antibody

A serum-free medium (SFM-MDM) without any animal-driven protein was developed for the production of recombinant antibody in suspension cultures of recombinant Chinese hamster ovary cell line, CS*13-1.00. The formulation of SFM-MDM was based upon the Iscove's modified Dulbecco's medium (IMDM), and Long™R 3IGF-1 and ferric citrate were supplemented instead of insulin and transferrin. Among various protein hydrolysates, yeast extract was selected as a serum-substituting additive for the improvement cell growth and productivity of recombinant antibody. Through about 3600 h of adaptation period from conventional SFM to SFM-MDM, integral of viable cell density and specific antibody productivity increased from 7.60 × 10 6 cells d and 19.14 μg (10 6 cells d) −1 to 7.79 × 10 6 cells d and 54.04 μg (10 6 cells d) −1, respectively, resulted in final antibody concentration of 420.98 mg l −1, which was about three times higher than that obtained from commercial medium, 486F. Also, supplementation of various vitamins including biotin, pyridoxal·HCl, and ascorbic acid was observed to enhance specific productivity, which led to high final antibody concentration. These results suggested the fundamental and effective strategy for development of a serum-free medium and to enhance cell growth and production of target proteins.

A novel control scheme for inducing angiostatin-human IgG fusion protein production using recombinant CHO cells in a oscillating bioreactor

In this study, a novel control scheme for inducing protein production using a recombinant CHO cell line in a BelloCell ® bioreactor was developed. This control scheme was applied in a simple regular semi-batch process. Production of angiostatin-human IgG fusion protein in a suspension recombinant CHO cell culture and a protein-free medium was used for this study. The bottom holding time (BH) was the sole operating variable to control the exposure time of the cells immobilized on the carriers to the air and allow the nutrient remained on the liquid film of the carriers to be consumed to a threshold level so that the cells can be arrested and promoted for protein production. In the cell cultures with various BH (1.5–90 min), final cell densities of 1.6–4.0 × 10 9 have been obtained in 20 days while total angiostatin-human IgG production of 228–388 mg have been harvested. In general, low BH will minimize the nutrient limitation and favor the cell growth, while high BH will restrict the nutrient and promote the production in this type of non-growth associated production systems. It was found that specific production rate was generally inversely proportional to the specific growth rate. In this case of study, BH of 30 and 60 min were found to be about 72% better than BH of 1.5 min and 35% better than BH of 9 and 90 min in term of the total angiostatin-human IgG production. In comparison to a conventional spinner flask study, a 3.8-fold increase of the total angiostatin-human IgG production was realized in a 35-day culture. This study illustrated that a simple method of using BH in a semi-batch process can effectively control the apparent nutrient concentration to the cells, and thus regulate the cell growth and protein production in a novel oscillating bioreactor.

Mechanisms of allosteric modulation at GABA B receptors by CGP7930 and GS39783: effects on affinities and efficacies of orthosteric ligands with distinct intrinsic properties

We determined the effects of the allosteric γ-aminobutyric acid B receptor modulators CGP7930 and GS39783 on binding and function of orthosteric ligands with distinct intrinsic properties. In radioligand binding (saturation or displacement) experiments, the affinities of a number of competitive antagonists were decreased by the modulators, with no change in receptor number. The binding curves of the partial agonist CGP47656 comprised a high and a low affinity component; the affinity of the former was increased by the allosteric agents. The maximal stimulation of GTP[γ] 35S binding via recombinant GABA B receptors by CGP47656 was increased 4-fold in the presence of 30 μM CGP7930 or GS39783. Two compounds known so far as “silent” competitive GABA B receptor antagonists, CGP35348 and 2-OH-saclofen, did not stimulate GTP[γ] 35S binding on their own, but became low efficacy partial agonists in the presence of the two modulators. The potency of GABA to inhibit the formation of cAMP induced by a forskolin analog in a recombinant CHO cell line expressing GABA B receptors was increased by the modulators. CGP35348 and 2-OH-saclofen, like CGP47656, were partial agonists on their own in this assay, and the allosteric modulators increased the potency as well as the efficacy of all three compounds. With CGP52432, there was a trend towards inverse agonism in the cAMP assay. These results show that the intrinsic properties of orthosteric ligands are highly dependent on the characteristics of the assay system used and that allosteric modulators are useful tools for elucidating these properties.

High-Level scu-PA Production by Butyrate-Treated Serum-Free Culture of Recombinant CHO Cell Line

The MGpUK-5 cell line, transformed with a single-chain urokinase-type plasminogen activator (scu-PA) minigene, generated mRNA transcripts and scu-PA titers corresponding to 65% or 86% of the amount generated before serum-free adaptation, despite significant loss of scu-PA gene copies during adaptation to serum-free culture. To further augment scu-PA production, a culture strategy employing sodium butyrate was explored. In 60-mL spinner flask cultures, sodium butyrate in the concentration range 1-10 mM allowed scu-PA production 2- to 3-fold higher than that in the negative control culture. Its productivity-enhancing activity was dependent on cell density in a range of 1-5 x 10(6) cells/mL, generating 72,200 +/- 8,100 IU/mL (480 +/- 50 mg/L) in 60-mL spinner flask cultures. To confirm this result, cells were grown to 4.4 x 10(6) cells/mL and treated with 5 mM sodium butyrate in a 2.5-L perfusion culture. The scu-PA titer increased more than 2-fold, and specific production rate of scu-PA increased 3-fold by this treatment. Overall, this perfusion culture gave rise to 1.7 x 10(8) IU scu-PA (1.1 g), comparable to total scu-PA production in a batch butyrate-treated culture performed at a 25-L bioreactor scale (1.3-3.5 g). Our results suggest that sodium butyrate treatment on high-density culture enables scu-PA production in gram quantities.

Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells

In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel((R)) disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (2.5x10(7) cell ml(-1) of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by -25% and -50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a -25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a -30% loss of productivity was obtained when the medium perfusion rate was further reduced to -50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of -25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufacturing operations.

Low-temperature pausing of cultivated mammalian cells

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.

The effects of phosphoramidon on the expression of human endothelin-converting enzyme-1 (ECE-1) isoforms.

Endothelin-1 (ET-1) is generated from big ET-1 by endothelin converting enzyme-1 (ECE-1). This process is inhibited by phosphoramidon through binding to the catalytic domain of ECE-1. There are four isoforms of human ECE-1 (ECE-1a, ECE-1b, ECE-1c and ECE-1d) which possess a conserved catalytic domain. Interestingly, a recent study has shown that in ECE-1b-transfected CHO cells phosphoramidon increases the expression and activity of ECE-1b. It is not known, however, whether phosphoramidon has similar effects on the expression of other ECE-1 isoforms. To address this point, we have established recombinant CHO cell lines that permanently express either human ECE-1a, ECE-1b or ECE-1c. Incubation of CHO/ECE-1a, -1b, and -1c with phosphoramidon (100 microM) for 16 hours markedly elevated the intracellular expression of ECE-1a and ECE-1b, but not ECE-1c protein, as indicated by Western blotting and immunocytochemistry. These increases appear to be due to inhibition of intracellular degradation of the protein because metabolic labeling followed by immunoprecipitation showed ECE-1a and ECE-1b proteins had prolonged half-lives in the phosphoramidon-treated cells. This is further supported by the finding that ECE-1 mRNA levels were unchanged following phosphoramidon treatment. Taken together, our results demonstrate that phosphoramidon differentially affects the expression of three human ECE-1 isoforms.

Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo (r) protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications.

An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter.

In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.

Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion.

On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment -capturing and rolling of leukocytes. A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor. In the smallest system 25.4 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 3.7 x 10(6) cells ml(-1). Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 x 10(7) cells ml(-1) in the 2 l bioreactor within 29 days. Large scale cultivation in a 100 l stirred tankbioreactor yielded 105.6 mg in three batches with a maximal viable cell density of 9.7 x 10(5) cells ml(-1) within 15 days. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration. Purificationwas performed by affinity chromatography with rProtein A. Integrity of the E-selectin-IgG protein was checked with SDS PAGE. Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively. Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively. A concentation of 18.8 and 37.5 mug ml(-1)was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.

Heparan Sulfate Glycosaminoglycans Are Receptors Sufficient To Mediate the Initial Binding of Adenovirus Types 2 and 5

Cell infection by adenovirus serotypes 2 and 5 (Ad2/5) initiates with the attachment of Ad fiber to the coxsackievirus and Ad receptor (CAR) followed by alpha(v) integrin-mediated entry. We recently demonstrated that heparan sulfate glycosaminoglycans (HS GAGs) expressed on cell surfaces are involved in the binding and infection of Ad2/5 (M. C. Dechecchi, A. Tamanini, A. Bonizzato, and G. Cabrini, Virology 268:382-390, 2000). The role of HS GAGs was investigated using extracellular soluble domain 1 of CAR (sCAR-D1) and heparin as soluble receptor analogues of CAR and HS GAGs in A549 and recombinant CHO cell lines with differential levels of expression of the two receptors and cultured to various densities. Complete inhibition of binding and infection was obtained by preincubating Ad2/5 with both heparin (10 microg/ml) and sCAR-D1 (200 microg/ml) in A549 cells. Partial inhibition was observed when heparin and sCAR-D1 were preincubated separately with Ad. The level of heparin-sensitive [(3)H]Ad2/5 binding doubled in sparse A549 cells (50 to 70,000 cells/cm(2)) with respect to that of cells grown to confluence (200 to 300,000 cells/cm(2)), in parallel with increased expression of HS GAGs. [(3)H]Ad2 bound to sparse CAR-negative CHO cells expressing HS GAGs (CHO K1). No [(3)H]Ad2 binding was observed in CHO K1 cells upon competitive inhibition with heparin and in HS GAG-defective CHO A745, D677, and E606 clones. HS-sensitive Ad2 infection was obtained in CAR-negative sparse CHO K1 cells but not in CHO A745 cells, which were permissive to infection only upon transfection with CAR. These results demonstrate that HS GAGs are sufficient to mediate the initial binding of Ad2/5.

High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium

A recombinant CHO cell line, CHO2DS, was immobilized on porous microcarrier Cytopore 1 and cultivated in 1 l modified Super-spinner and 2 l stirred tank bioreactor with the perfusion of a low-cost chemically defined protein-free medium DF6S. CHO2DS cells could enter into the inner space and grew both in the inner space and on the surface of Cytopore 1 in DF6S and produced prothrombin at 22 mg l−1 after 10 days. From a seeding density of 5.7 × 105 cells ml−1, the highest viable cell density of CHO2DS was 1.12 × 107 cells ml−1.

Pilot production of u-PA with porous microcarrier cell culture.

A recombinant DNA CHO cell line secretingurokinase-type plasminogen activator (u-PA) wascultivated with Cytopore cellulose porousmicrocarriers in a 30l Biostat UC stirred tankreactor. After 26 days of culture, using a spinfilter toretain cells in bioreactor, the cell density couldreach 1.33 x 10(7) ml(-1). The maximal u-PAactivity in supernatant was 7335 IU.ml(-1), and204l supernatant containing 7.1 g u-PA was harvested.After 100 days of culture with 0.1% fetal bovineserum medium, a modified cell retention system whichcan be washed-out backward, substituted thespinfilter to prevent filter clogging. The maximalcell density was over 10(7) ml(-1), the maximalu-PA activity in supernatant reached 6250IU.ml(-1), and 1604l supernatant containing about51 g u-PA was harvested. Compared to perfusionculture, batch medium-replaced culture could raiseutilizing efficiency of the medium, increase cell specificproductivity and improve the quality of the product which wasnot steady in a 37 degrees C environment. Cells can movefrom seed porous microcarriers occupied by cells tovacant microcarriers spontaneously, withouttrypsinization, and continue to grow until all microcarriers contained cells. It shows that Cytoporeporous microcarriers are very useful and convenient toscale up cultivation step by step.

A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin

A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 × 105 cells ml−1, CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 × 106 cells ml−1 with an accumulated prothrombin concentration of 16.7 mg l−1 after 6 days in culture.

Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 mum spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale.

Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 mum spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mumspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 mum was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s(-1) (141 rpm) - furtherenhancement of spinfilter rotation velocity up to0.56 m s(-1) (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s(-1)(35 rpm) and 1.26 m s(-1) (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.

GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode.

This study correlates the fluorescent signal from stable recombinant CHO cell lines expressing the green fluorescent protein (GFP) at high levels with biomass or cell number, extending the use of fluorescent proteins to applications and assays where cell growth rates are important. Using a standard fluorometer, growth of these cells can be quantified noninvasively in multiwell plates, and because signals are obtained without preparation, the same culture samples can be measured repeatedly. Even with a small relative change in biomass, the specific growth rate can be determined in a few hours. The dynamics of cell populations can now be studied with high sensitivity, low error rate, and minimum sample preparation.

Changes during subclone development and ageing of human antibody-producing recombinant CHO cells

Some of the problems encountered with human or human-mouse heterohybridomas, such as low growth rates and high serum requirements, have led to the increased use of recombinant cell lines for production of human antibodies. To evaluate the suitability of such alternative cell lines for the production of human antibodies we have analysed several subclones with differing specific production rates of a recombinant CHO cell line. Gene copy number and site of chromosomal integration for the light and heavy chain and the dhfr gene were determined by in-situ hybridisation. Specific mRNA content was analysed by Northern blot. In addition the intracellular content in light and heavy chain was measured by flow cytometry and the specific secretion rates were determined. The stability of gene expression was followed in the highest producing subclone for over a year. As previously seen in heterohybridoma cells a high expression rate of light chain is beneficial in speeding up secretion rates of whole antibody. When grown in the presence of G418 and methotrexate the amplified gene copies in the genome of recombinant CHO cells were stable over more than 100 passages. However, the expression of light chain, and with it the secretion rate, decreased with time. The low intracellular concentration of light chain resulted in accumulation of heavy chain in the endoplasmic reticulum due to retention by chaperones. The specific secretion rate decreased by 50% after 100 passages. When no G418 or methotrexate were present 75% of the gene copies were lost after 100 passages.

High density and scale-up cultivation of recombinant CHO cell line and hybridomas with porous microcarrier Cytopore.

Using porous microcarrier Cytopore and a low-serum medium supplement BIGBEF-3, we have successfully cultivated recombinant CHO cell line CL-11G producing prourokinase and hybridomas producing anti-prourokinase monoclonal antibody in Celligen 1.5 or 5 L bioreactor. The cell density obtained ranged from 1 to 2 x 107 cells mL-1. The yields of prourokinase and monoclonal antibody increased with increasing cell density. As the cells could spontaneously release from and reattach to porous microcarriers, it was very easy to scale-up the cultivation. Thus the bead to bead cell transfer method has been used to scale up the cultivation of CL-11G cells to a 20 L reactor-scale for the pilot production of prourokinase, and also to scale-up the culture of hybridomas for the production of monoclonal antibody for the purification of prourokinase.

The role of the cell cycle in determining gene expression and productivity in CHO cells.

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.

Generation of mice expressing the human glucagon receptor with a direct replacement vector.

The process of evaluating the in vivo efficacy of non-peptidyl receptor antagonists in animal models is frequently complicated by failure of compounds displaying high affinity against the human receptors to show measurable affinity at the corresponding rodent receptors. In order to generate a suitable animal model in which to evaluate the in vivo activity of non-peptidyl glucagon receptor antagonists, we have utilized a direct targeting approach to replace the murine glucagon receptor with the human glucagon receptor gene by homologous recombination. Specific expression of the human glucagon receptor (GR) in the livers of transgenic mice was confirmed with an RNase protection assay, and the pharmacology of the human GRs expressed in the livers of these mice parallels that of human GR in a recombinant CHO cell line with respect to both binding of 125I-glucagon and the ability of glucagon to stimulate cAMP production. L-168,049, a non-peptidyl GR antagonist selective for the human GR shows a 3.5 fold higher affinity for liver membrane preparations of human GR expressing mice (IC50 = 172 +/- 98 nM) in the presence of MgCl2 in marked contrast to the measured affinity of the murine receptor (IC50 = 611 +/- 197 nM) for this non-peptidyl antagonist. The human receptors expressed are functional as measured by the ability of glucagon to stimulate cAMP production and the selectivity of this antagonist for the human receptor is further verified by its ability to block glucagon-stimulated cyclase activity with 5 fold higher potency (IC50 = 97.2 +/- 13.9 nM) than for the murine receptor (IC50 = 504 +/- 247 nM). Thus we have developed a novel animal model for evaluating GR antagonists in vivo. These mice offer the advantage that the regulatory sequences which direct tissue specific and temporal expression of the GR have been unaltered and thus expression of the human gene in these mice remains in the normal chromosomal context.