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PubMed2006

Natural isolates of Saccharomyces cerevisiae display complex genetic variation in sporulation efficiency.

Sporulation is a well-studied process executed with varying efficiency by diverse yeast strains. We developed a high-throughput method to quantify yeast sporulation efficiency and used this technique to analyze a line cross between a high-efficiency oak tree isolate and a low-efficiency wine strain. We find that natural variation in sporulation efficiency mirrors natural variation in higher eukaryotes: it shows divergence between isolated populations, arises from loci of major effect, and exhibits epistasis. We show that the lower sporulation efficiency of the wine strain results from a failure to initiate sporulation, rather than from slower kinetics of meiosis and spore formation. The two strains differentially regulate many genes involved in aerobic respiration, an essential pathway for sporulation, such that the oak tree strain appears better poised to generate energy from this pathway. We also report that a polymorphism in RME1 that affects sporulation efficiency in laboratory strains also cosegregates with significant phenotypic differences in our cross of natural isolates. These results lay the groundwork for the study of variation in sporulation efficiency among natural isolates of yeast.

Genetic Variation↗

PubMed1997

Gene transfection by cationic liposomes: comparison of the transfection efficiency of liposomes prepared from various positively charged lipids.

We compared the transfection efficiency of four types of positively charged liposomes composed of (i) N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylethanolamine (DOPE) (1:2:2 molar ratio); (ii) 3 beta [N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and DOPE (3:2 molar ratio); (iii) dimethyldioctadecylammonium bromide (DDAB) and DOPE (1:2.2 molar ratio); (iv) N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and DOPE (1:1, w/w; lipofectin). Luciferase gene was used as a reporter gene. Among the cationic lipsomes used, the liposomes composed of TMAG, DOPE and DLPC showed a much higher efficiency of plasmid DNA entrapment than the other cationic liposomes tested. In the absence of serum, the cationic multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) composed of TMAG, DOPE and DLPC gave highly efficient transfection. On the other hand, MLV, dehydration-rehydration vesicles (DRV), and SUV liposomes prepared with the mixtures of DC-Chol and DOPE showed similar levels of transfection efficiency. However, the cationic liposomes composed of DDAB and DOPE showed inferior efficiency, whether in the form of DRV, SUV or MLV. The transfection efficiency of lipofectin was also low. In the presence of serum, on the other hand, a considerable (about 30-50%) amount of transfection activity was still observed at 10% fetal calf serum in the cationic MLV and SUV composed of TMAG, DOPE and DLPC. Cationic MLV, composed of TMAG, DOPE and DLPC, Cationic MLV, composed of TMAG, DOPE and DLPC, can transfect plasmid DNA, not only in the adherent cell lines but also, in the suspension cell lines. These findings indicate that the transfection efficiency of cationic liposomes is affected by the lipid composition, the type of liposome, or the presence or absence of serum. They also indicate that the cationic liposomes containing TMAG, DOPE and DLPC are efficient vectors for gene transfer into cells.

Animals↗

PubMed2004

High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol.

Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast. We report here that pretreatment of P. pastoris with 0.1 M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 150-fold. DTT alone enhanced the transformation efficiency up to 20-fold, but LiAc alone had little effect. Cultures grown to 1.15-2.6 A at 600 nm had higher transformation efficiencies than younger or older cultures. A cell concentration of 10(10)/mL gave the highest efficiencies. Digestion of pPIC9K within the AOX1 gene with Sacl gave efficiencies approximately 30 times higher than digestion in other genes with other enzymes. Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kV, 25 microF, and 186 omega. The transformation efficiency at optimal conditions reached 4 x 10(6) transformants/microgram DNA with pPIC9K. A maximum of 2.6 x 10(5) transformants was produced when 1 microgram of pPIC9K DNA was used.

Acetates↗

PubMed1994

Economic incentives and disincentives for efficient prescribing.

Efficient prescribing is a controversial concept when defined in economic terms. Such terms imply that neither the best nor the cheapest drug may be efficient. Efficiency depends on patient evaluations of outcomes--an essential, yet still underdeveloped, field of inquiry. Encouraging prescribing efficiency (once it is clear what efficient treatments are) may require the use of incentives, (economic and otherwise). Although physicians do react to economic incentives, encouraging them to also act as good economists rather than only as good healthcare providers may be difficult since economic efficiency goals may conflict with pure medical goals. While it is conceptually reasonable to assume that economic incentives will accomplish behavioural change, other (noneconomic) incentive mechanisms may appear less controversial. Since it is the result, not the process, that is important, a mix of incentives to achieve efficient solutions is suggested. However, further investigation into both health and economic outcomes research and debate on proper efficiency goals should precede the discussion of optimal incentives.

Cost-Benefit Analysis↗

PubMed1990

Biological efficiency to weaning and to slaughter of crossbred beef cattle with different genetic potential for milk.

Biological efficiency of beef production to weaning and to slaughter was estimated in three groups of cattle reasonably similar in growth and mature size but different in the amount of milk available to the calves (low (L) = Hereford x Angus, medium (M) = Red Poll x Angus and high (H) = Milking Shorthorn x Angus). Efficiency was defined as the ratio of estimated kilograms of calf weight weaned or carcass weight produced by a herd of 100 first-cross cows to estimated metabolizable energy (ME) intake by the cows and preweaning non-milk ME intake by the calves or preweaning non-milk and postweaning ME intake by the calves. Efficiencies were estimated assuming observed and average reproductive rates, and using observed energy requirements for maintenance, as well as the same requirements for maintenance in the M and H groups. With the observed reproductive rates and requirements for maintenance, efficiencies to weaning and to slaughter were 28.1, 27.4 and 27.4 g weaning weight per Mcal ME and 22.0, 20.6 and 20.3 g carcass weight per Mcal ME for the L, M and H groups, respectively. With average requirements for maintenance, efficiencies were 28.2 and 27.5 to weaning and 20.8 and 20.4 to slaughter for the M and H groups, respectively. Under equal reproductive rates and observed maintenance requirements, efficiencies were 28.3, 27.2 and 27.7 to weaning, and 22.1, 20.6 and 20.6 to slaughter for the L, M and H groups, respectively. With average maintenance requirements, efficiencies were 27.5 and 27.4 to weaning and 20.7 and 20.5 to slaughter for the M and H groups, respectively. Across the production output and input assumption scenarios, the L group consistently was the most efficient, especially when evaluated at slaughter of calves.

Age Factors↗

PubMed1999

Novel insight into the control of litter size in pigs, using placental efficiency as a selection tool.

Chinese Meishan pigs produce three to five more pigs per litter than less-prolific U.S. or European pig breeds as a result of a markedly decreased placental size and an increased pig weight: placental weight ratio (placental efficiency). We hypothesized that as a result of their intense selection for prolificacy, the Chinese had indirectly selected for a smaller, more efficient placenta in the Meishan breed. The goals of this study were to determine whether 1) significant variation in placental size and efficiency existed within our population of purebred Yorkshire pigs and 2) selection of pigs (boars and gilts) based on clear differences in placental size and efficiency would affect litter size. There was significant (approximately threefold) variation in placental efficiency in our herd of Yorkshire pigs, and marked (approximately twofold) variation existed within individual litters. We then selected pigs (boars and gilts) that had either a higher (A Group) or lower (B Group) than average placental efficiency. Although the birth weights of selected A Group pigs were similar to those of the B Group pigs, they had markedly smaller placentae. Males from each group (A or B) were bred to the females of the same group, and farrowing data were collected from parities 1 and 2. In both parities, A Group females farrowed more live pigs per litter than did B Group females (12.5 +/- .7 vs 9.6 +/- .5, P < .05). Although A Group pigs were on average approximately 20% lighter than B group pigs (1.2 +/- .1 vs 1.5 +/- .1 kg, P < .05), their placentae were approximately 40% lighter (250 +/- 10 vs 347 +/- 15 g, P < .01), resulting in a marked increase in placental efficiency. The results of this study suggest that selection on placental size and efficiency may provide a valuable tool for optimizing litter size in commercially important pig breeds.

Animal Husbandry↗

PubMed1983

Phenotype x nutritional environment interactions in forage intake and efficiency of Angus cows grazing fescue-legume or fescue pastures.

Forage intake, milk production, calf growth and efficiencies of milk production and calf growth were estimated for 198 lactations of 91 mature Angus cows grazing fescue-legume or fescue pastures. Cows calved January through March and calves were weaned in October in each of the 5 yr of the experiment. Cows grazing fescue-legume consumed 1.7 kg/d more (P less than .01) dry matter (DM) that was 4.6 percentage units more (P less than .01) digestible than cows grazing fescue. Cows grazing fescue-legume produced .4 kg/d more (P less than .01) milk and weaned 23.8 kg more (P less than .01) calf than cows grazing fescue. This increased production required an additional intake/cow-calf unit of 2.3 kg of digestible DM for each additional kilogram of calf weaned. Cows grazing fescue-legume were less efficient in calf production at least partially because they gained 33.3 kg more (P less than .01) weight and 1.82 mm more (P less than .01) fat cover at the 12th rib during lactation than cows grazing fescue. Cows producing relatively large amounts of milk consumed more digestible DM, weaned heavier calves, and were more efficient producers of milk and weaned calf regardless of pasture type grazed. The influence of milk production on these variables was more dramatic for cows grazing fescue than for those grazing fescue-legume. Cow type favoring maximum conversion of digestible DM intake by cow and calf to calf gain was different for the two types of pasture. For cows grazing fescue, those having either small amounts of fatness or having small structural sizes, regardless of milk production, were the most efficient. Structural size was not related to efficiency for cows grazing fescue-legume. Fatness had little influence on efficiency of low milking cows. The relationship of structural dimension and efficiency for cows grazing fescue was due to interrelationships of structural dimension, change in cow weight and fatness during lactation, and calf weaning weight: cow weight ratio. Relationships between efficiency and fatness could not be attributed to these variables.

Animal Feed↗

PubMed1987

Simulated efficiency of range beef production. I. Growth and milk production.

A revised version of the Texas A&M University Beef Cattle Production Model was used to simulate the effects of growth, milk production and management system on biological and economic efficiency of beef production in a northern plains, range environment. Animals varying in genetic potential for birth weight (BWA), yearling weight (YW), mature weight (WMA) and milk production (PMA) were simulated under both a weanling system of management (weaned calves custom-fed in the feedlot) and a yearling system (calves wintered on the ranch, then custom-fed after their second summer). The yearling system of management was biologically less efficient, but economically more efficient than the weanling system due primarily to heavier slaughter weights of fed animals. The advantage of the yearling system was most apparent for smaller genotypes. Herd efficiency improved with decreased BWA and increased YW, but changed little when WMA was varied while other growth traits were held constant. Increased PMA was favored for production of live weight at weaning and for production of slaughter product when feedlot costs were high. Increased PMA was not favored when feed costs for the cow herd were high. Economic weights generated from the simulation indicated the importance of selection for rapid early growth followed by selection for lighter birth weight. While larger genotypes were generally favored in this study, optimal cow size depended on economic conditions. Larger types were more biologically efficient and more economically efficient using standard costs, but medium- and small-size cattle were more efficient when feedlot costs were high. Small cattle were least efficient when feed costs for the cow herd were high.

Animal Husbandry↗

PubMed2002

Dairy herd management practices that impact nitrogen utilization efficiency.

Improving the efficiency of feed N utilization by dairy cattle is the most effective means to reduce nutrient losses from dairy farms. The objectives of this study were to quantify the impact of different management strategies on the efficiency of feed N utilization for dairy farms in the Chesapeake Bay Drainage Basin. A confidential mail survey was completed in December 1998 by 454 dairy farmers in PA, MD, VA, WV, and DE. Nitrogen intake, urinary and fecal N, and efficiency of feed N utilization was estimated from survey data and milk analysis for each herd. Average efficiency of feed N utilization for milk production by lactating dairy cows (N in milk/N in feed x 100) was 28.4% (SD = 3.9). On average, farmers fed 6.6% more N than recommended by the National Research Council, resulting in a 16% increase in urinary N and a 2.7% increase in fecal N. Use of monthly milk yield and component testing, administration of bovine somatotropin (bST), and extending photoperiod with artificial light each increased efficiency of feed N utilization by 4.2 to 6.9%, while use of a complete feed decreased efficiency by 5.6%. Increased frequency of ration balancing and more frequent forage nutrient testing were associated with higher milk production, but not increased N utilization efficiency. Feeding protein closer to recommendations and increasing production per cow both contributed to improving efficiency of feed N utilization.

Animal Nutritional Physiological Phenomena↗

PubMed2004

Relationships of efficiency to reproductive disorders in Danish milk production: a stochastic frontier analysis.

Relationships of various reproductive disorders and milk production performance of Danish dairy farms were investigated. A stochastic frontier production function was estimated using data collected in 1998 from 514 Danish dairy farms. Measures of farm-level milk production efficiency relative to this production frontier were obtained, and relationships between milk production efficiency and the incidence risk of reproductive disorders were examined. There were moderate positive relationships between milk production efficiency and retained placenta, induction of estrus, uterine infections, ovarian cysts, and induction of birth. Inclusion of reproductive management variables showed that these moderate relationships disappeared, but directions of coefficients for almost all those variables remained the same. Dystocia showed a weak negative correlation with milk production efficiency. Farms that were mainly managed by young farmers had the highest average efficiency scores. The estimated milk losses due to inefficiency averaged 1142, 488, and 256 kg of energy-corrected milk per cow, respectively, for low-, medium-, and high-efficiency herds. It is concluded that the availability of younger cows, which enabled farmers to replace cows with reproductive disorders, contributed to high cow productivity in efficient farms. Thus, a high replacement rate more than compensates for the possible negative effect of reproductive disorders. The use of frontier production and efficiency/inefficiency functions to analyze herd data may enable dairy advisors to identify inefficient herds and to simulate the effect of alternative management procedures on the individual herd's efficiency.

Animals↗

PubMed1989

Genetic parameters of estimated net energy efficiencies for milk production, maintenance, and body weight change in dairy cows.

Net efficiencies of converting intake energy into energy for maintenance, milk production, and body weight change in a lactation were estimated for each of 79 Holstein cows by a two-stage multiple regression model. Cows were from 16 paternal half-sib families, which each had members in at least two of the six herds. Each cow was recorded for milk yield, net energy intake, and three efficiency traits. These were analyzed in a multitrait model containing the same 14 fixed subclasses of herd by season by parity and a random factor of sires for each of the five traits. Restricted maximum likelihood estimates of sire and residual (co)variance components were obtained by an expectation maximization algorithm with canonical transformations. Between milk yield and net energy intake, net energy efficiencies for milk yield, maintenance, and body weight change, the estimated phenotypic correlations were .36, -.02, .08, and -.06, while the genetic correlations were .92, .56, .02, and -.32, respectively. Both genetic and phenotypic correlations were zero between net energy efficiency of maintenance and that of milk yield and .17 between net energy efficiency of body weight change and that of milk yield. The estimated genetic correlation between net efficiency for lactation and milk yield is approximately 60% of that between gross efficiency and milk yield. With a heritability of .32 equivalent.49, net energy efficiency for milk yield may be worth consideration for genetic selection in certain dairy cattle populations.

Animals↗

PubMed1998

Efficiency of nutrient use and relationship to profitability on dairy farms.

As cows eat more feed to support higher milk production, the proportion of digested energy that is captured in milk increases. In contrast, as cows consume more feed, digestive efficiency decreases, but the magnitude of depression in digestibility is not characterized for high producing cows. Despite the digestibility depression, biological efficiency increases considerably as Holstein cows produce more milk up to 15,000 kg/yr. Above 15,000 kg/yr, the gain in biological efficiency per unit of increase in milk production is expected to approach 0. At 21,000 kg of milk/yr, approximately 25% of the gross energy consumed by the cow during the first 5 yr of life likely would be captured as milk, conceptus, or body tissues; further gains in biological efficiency are not likely without major advances in feed digestion. Although feeds generally cost more as cows are fed for higher milk production, increased productivity also enhances profitability, partly because of increased efficiency but also because fixed costs are decreased relative to total costs. This relationship between productivity and profitability is expected to continue up to > 21,000 kg/yr per cow. Other issues related to the efficiency of nutrient use will become more important in the future, namely, efficiency of the use of feeds that can also be consumed by humans, efficiency of use of tillable land, and minimization of nutrient losses to the environment.

Animal Nutritional Physiological Phenomena↗

PubMedDate not supplied

Enhancement of gene transfer efficiency into human cancer cells by modification of retroviral vectors and addition of chemicals.

Retroviral vectors have recently experienced limited use in cancer gene therapy mainly due to poor transduction efficiency. To overcome this drawback, we attempted to enhance the transduction efficiency by employing different retroviral packaging cell lines and chemical additives. The retrovirus from the PG13 packaging cell line gave mostly higher or similar transduction efficiencies in a variety of human cancer cell lines compared to the retrovirus from the PA317, Bing, or FLYRD18 packaging cell line. A cationic liposome, especially Lipofectamine, significantly enhanced the transduction efficiency of a retrovirus. However, the retrovirus derived from the PG13 cell line could not infect the murine cell line efficiently even after Lipofectamine treatment. Furthermore, chloroquine did not improve the transduction efficiency regardless of the presence of chemical additives. These results, therefore, suggested that the transduction efficiency of a retrovirus in human cancer cells can certainly be improved when a proper packaging cell line is chosen. In addition, this study implied that Lipofectamine is a superb additive to enhance the transduction efficiency of a retrovirus via a specific virus envelope protein-receptor interaction for virus entry, and that receptor-mediated endocytosis does not seem to be the leading route of virus delivery to liberate a virus genome.

3T3 Cells↗

PubMed2000

An evaluation of flare combustion efficiency using open-path Fourier transform infrared technology.

Open-path Fourier transform infrared (OP-FTIR) technology was used to evaluate the combustion efficiency of a flare for comparison to several combustion models. Most flares have been considered an effective method for destroying organic compounds and anything that burns. As the Btu content of the flare gas is reduced, the combustion efficiency may also be reduced. Recent studies have suggested that lower Btu flares may have efficiencies as low as 65%. In addition, models have been developed to predict the effect of wind speed and stack discharge velocity on the combustion efficiency. This study was conducted on a low-Btu flare gas that is primarily CO. While the models would predict efficiencies as low as 30%, the sampling using OP-FTIR showed most combustion efficiencies well above 90%. Three methods were used to track combustion efficiency: monitoring the ratio of CO to CO2, monitoring the ratio of CO to tracer gas, and dispersion modeling. This study was complicated by the presence of two flare stacks, thus two tracer gases were used--SF6 and CF4. A method was developed for distinguishing between the two stacks and quantifying the efficiency in each stack.

Air Pollution↗

PubMed1995

Effects of 20 days bed rest on mechanical efficiency during upright cycling and leg muscle mass in young males.

Delta efficiency defined as increase in work over the corresponding increase in energy liberation (delta work/delta energy) may be used to express the efficiency of working muscles under standard conditions where work is performed with similar changes in muscle length, identical pedal revolution frequencies, and contraction-to-relaxation ratios. The Delta efficiency is probably the most valid measure of the efficiency of muscular work, so it may be influenced by the difference in distribution and/or density of muscle fiber types in exercising muscles. It has been reported that after bed rest of 7-14 days, not only maximum oxygen uptake (VO2 max) but also oxygen uptake (VO2) at 3-min submaximal upright exercise decreased. However, the decrease might be apparent, and the mechanical efficiency might be unchanged. On the other hand, muscle mass of bicycling legs was decreased after continuous horizontal bed rest of 10 days and 20 days. Because the decreased muscle mass is probably related to decrease in the density of the slow twitch muscle fibers (ST-fiber), the decrease in submaximal VO2 during bicycle exercises after bed rest may result from a decrease in ST-fiber mass. Therefore, it could be hypothesized that the mechanical efficiency should increase during upright exercise because of the relative increase in amount of more efficient fast twitch muscle fibers (FT-fiber) than ST fibers in exercising muscles. The purpose of the present study was to investigate whether delta efficiency in working muscle is influenced by the decrease in muscle mass after 20 days horizontal bed rest in young males.

Adult↗

PubMed2001

Efficient intracellular delivery of an exogenous protein GFP with genetically fused basic oligopeptides.

Several oligopeptides, derived from certain proteins, translocate as a form fused to small molecules or exogenous proteins across the plasma membrane into cells. Some of these oligopeptides, the so-called protein-transduction domains (PTDs), contain a high proportion of basic residues. The translocation of some of these basic PTDs, such as oligoarginines, has been studied as chemically fused forms to other organic compounds. In this study, we also tested to determine whether or not oligoarginines, when fused genetically to an exogenous protein such as GFP, are also able to translocate efficiently across the plasma membrane. The oligoarginine Rn (n = 5,6,7,8,9)-GFP fusion proteins were translocated quite efficiently, and the transduction efficiency increased in proportion to the number of arginine residues. However, the cellular uptake of the oligolysine-GFP fusion proteins was less efficient than that of the corresponding oligoarginine-GFP fusion proteins. When fused to GFP, the translocation efficiency of R5 was similar to that of Tat(49-57)(RKKRRQRRR). This finding suggests that the arginine homo-oligopeptide is more efficient than other PTDs which contain a mixture of basic residues. On the other hand, both the K9- and Tat(49-57)-GFP fusion proteins were transduced with similar efficiencies. It appears that basic oligopeptides may be useful for the efficient translocation of diverse exogenous proteins as genetically fused forms.

Amino Acid Sequence↗

PubMed2001

Scale, Efficiency and Organization in Norwegian Psychiatric Outpatient Clinics for Children.

BACKGROUND: It is generally believed that 5 percent of the population under 18 years is in need of specialist psychiatric care. In 1998, however, services were delivered to only 2.1 percent of the Norwegian population. Access to services can be improved by increasing capacity, but also by increasing the utilization of existing capacity. Changing financial incentives has so far not been considered. Based on a relatively low number of registered consultations per therapist (1.1 per therapist day) the ministry has stipulated that productivity should increase by as much as 50 percent. AIMS OF THE STUDY: Measuring productivity in psychiatric care is difficult, but we believe that studies of productivity should be an important input in policy making. The aim of this paper is to provide such an analysis of the productive efficiency of psychiatric outpatient clinics for children and youths, and in particular to focus on three issues: (i) is an increase in productivity of 50 percent a realistic goal, (ii) are there economies of scale in the sector, and (iii) to what extent can differences in productivity be explained by differences in staff-mix and patient-mix? METHODS: We utilize an approach termed Data Envelopment Analysis (DEA) to estimate a best-practice production frontier. The potential for efficiency improvement is measured as the difference between actual and best-practice performance, while allowing for trade-offs between different staff groups and different mixes of service production. The DEA method gives estimates of efficiency and productivity for each clinic without the need for prices, and thus avoids the pitfalls of partial productivity ratios. The Kolmogorov-Smirnov statistic is used to compare efficiency distributions, providing tests of variable specification and scale properties. RESULTS: Based on 135 observations for the years 1997 to 1999, the tests lead to a model with two inputs, two outputs and variable returns to scale. The outputs are number of hours spent on direct and indirect interventions, while neither the number of interventions nor the number of patients was found to be significant. The inputs are the number of university-educated staff and other staff, but disaggregation of the latter group was not significant. The average of estimated clinic efficiencies is 71%. The mean productivity is 64%, but many large clinics have considerably lower performance due mainly to scale inefficiency. DISCUSSION: There seems to be considerable room for improved performance in these clinics. It is interesting that the potential is not that far from the officially stipulated goal of 50% increased productivity. Staff composition does matter for clinic performance, but the different groups do not have significantly different marginal productivities, indicating a lack of ability to utilize specialized skills. It should be noted that these results to some extent depend on the assumptions that medical practice is efficient, and that the available data accurately captures the activities of the clinics. IMPLICATIONS FOR FURTHER RESEARCH AND HEALTH POLICY: More appropriate outcome measures, e.g. global assessment of functioning scores (GAF), will soon be available and will improve the policy value of this type of analysis, as will a more refined data set with information about the number of personnel in training positions. The analyses in this paper indicate that a lack of consensus on the issues of who should be treated, how they should be treated and by whom results in large variations in productive efficiency. These issues are being debated in Norway, and it should be interesting to see whether this in itself leads to higher efficiency or whether a change in the incentive structure will be needed.

Journal Article↗

PubMed2003

[Rapid and efficient expression of foreign genes in mammalian cells by baculovirus vectors].

The baculovirus insect cell expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, reports have described that recombinant baculoviruses can transduce a broad spectrum of primary and established mammalian cells, which shows the baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. In this report, we further research the modification of baculovirus vector and the way to deliver exogenous gene into mammalian cells. On the base of Bac-to-Bac baculovirus insect cell expression system, two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) were constructed containing different direction of CMV promoters which controll the expression of a reporter gene (EGFP). We found that CMV promoter could direct expression of reporter gene in Sf9 cells with relatively low efficiency. The culture supernatant of Sf9 cells which have been infected by the recombinant baculoviruses for four days were collected and the titers of the viruses in culture supernatant were determined by plaque assay on Sf9 cells. The HepG2 cells, an human hepatocellular carcinoma cell line, were directly incubated with the collected culture supernatant which contains the recombinant baculoviruses for 8 hours in 37 degrees C CO2 incubator (moi = 100). Twenty-four hours post transduction the efficiencies of gene-transfer and expression were analyzed by flow cytometry (FCM) which detect the green fluorescence of individual cells. Results show that these two recombinant baculoviruses have similar gene-transfer and expression efficiency in HepG2 cells, which means the direction of CMV promoters has no effects on reporter gene expression. The optimal transduction conditions of incubating the mammalian cells with the culture supernatant of Sf9 cells infected by recombinant baculoviruses for four days were determined by FCM assay in HepG2 cells. The HepG2 cells inoculated in 24-well plate (5 x 10(4)/well) were incubated with the culture supernatant (BacV-CMV-EGFPA, 1.2 x 10(7) pfu/mL) serially diluted by DMEM culture medium containing 10% FBS and the transduction times ranged from 1 to 24 hours. Twenty-four hours post transduction the efficiencies of gene-transfer and expression were analyzed by FCM. Results show that incubating the target cells with the 1:1 diluted culture supernatant (moi = 50) for 12 hours in 37 degrees C CO2 incubator would achieve the highest infection and expression efficiency with the least impairment on cell viability. We compared the gene-transfer and expression efficiency of recombinant baculovirus in HepG2 and CV1 cells with lipofectAMINE and recombinant retrovirus system, results show that under the similar conditions the recombinant baculovirus could achieve the highest gene-transfer and expression efficiency than the other two systems. So we can draw a conclusion that directly incubating the mammalian cells with the culture supernantant of the infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign gene in mammalian cells.

Animals↗