Bentley 23 Tubular Bioreactor for Probing Baculovirus Infection and Protein Production Hsuan-Chen Wu, Yu-Chen Hu and William E. Finally, during the decline phase the cell death rate exceeds the cell growth rate and the viable cell number declines. A sufficient quantity of the adapted cells should be cryopreserved to initiate future suspension cultures see Subheading 3. Leave the best performing individuals unchanged and allow the others to mate, in performance order, with different individuals. Place on a warm surface under 65°C or simply in the flow of air in a laminar flow hood. Loosen the caps on the medium and new culture flask s. Polyhedrin Gene Locus Expression Vectors The first transfer vectors to be developed for baculovirus expression systems were based on the polyhedrin gene promoter.
Plot the cell density vs glucose concentration data for the exponential growth data for times from —72 to 0 h pi as shown in Fig. Several early viral promoters, including those of 39K, the immediate-early gene ie-2 formerly ie-n , p143 and p35, have been shown to be stimulated by cis-linkage to hrs 68,73,75,80—82. . If lab-prepared materials are being used, solutions should be prepared fresh each time the isozyme technique is employed. Virus particles are occluded within polyhedrin protein P or granulin G. Currently, one is limited to three BaculoDirect constructs, i. So, human life will be harmonious and full of peace.
A major protein of the capsid, vp39, has also been identified. While holding it 192 Lynn inside the hood, peel down the protective wrapper on the end containing the cotton plug 5—10 cm. This arrangement has been implicated in the temporal regulation of these genes. If it falls to less than 80%, then a decrease of agitation is in order until growth continues and viability exceeds 80%. Therefore, one must either account for oxygen being transferred from the headspace generally not an easy task or eliminate oxygen in the head space e. Lynn prudent to put them through a decontamination process such as autoclaving for the peace of mind of refuse workers. There are also methods for the immunodectection and production of immunological reagents and the analysis of biochemical properties, such as subunit size, lipase kinetics, enzyme immobilization, and ligand interactions.
Insert the plugged end into the mechanical pipettor and pull the wrapper the rest of the way off the pipet, being careful not to touch the pipet to anything. Introduction The last 20 yr have seen baculoviruses maintain a reputation for producing high yields of recombinant proteins in insect cells. Example data for Sf-9 cell growth is given in Table 1. Finally, methods are discussed for storing cell cultures and issues with virus stocks of which the baculovirologist should be aware. Plot of cell density vs glucose concentration in the exponential phase for Sf-9 cell culture in serum-free medium.
The alkaline environment of the insect midgut causes the polyhedra to dissolve releasing the embed- ded virions. In this particular case the cell cultures did not suffer from nutrient depletion. Many of the nutrients and byproducts can be determined using a bioprofile monitor by following the manufacturer directions. On day 3 postplanting aspirate spent medium from one side of the monolayer and refeed the culture with fresh serum-free medium gently introduced to the side of the T-flask. Adding to the expense of spinner flasks, the author has found that spinner flask parts need to be replaced periodically as autoclaving seems to accelerate their wear out rate.
The baculovirus expression system is based on introduction of the foreign gene into nonessential regions of the viral genome through allelic replacement. Davis 14 Production Of Virus-Like Particles For Vaccination Christine M. Plot the glucose concentration vs time as given in Fig. Cite this protocol as: King L. A total volume of 4—5 mL is normally used in a 25-cm2 flask.
In such a case, these aggregates can be disrupted by pipetting into and out of a Pasteur pipet. The system is also amenable to parallel processing of many recombinant viruses using manual or automated methods. Simply incubating an aliquot of the medium is an adequate sterility testing for microbial contamination in most cases. Some indication of the proper split ratio should be provided by the source of the specific cell line and a range of split ratios should be used during the initial subcultures. Set the probability for crossover mating to occur at 95% and the mutation rate at 1% per bit according to ref. So, you will neverdo same mistakes again and again. Eventually, the caterpillar stops feeding and undergoes several rapid physiological changes.
Lynn Summary The various methods for maintaining a. This material can be stored at —20°C for several months. Calculate the natural logarithm ln of the exponential phase cell densities between 39. The author prefers using shaker flasks instead of spinner flasks to grow and infect insect cells for a number of reasons. Follow steps 1—7 of shake-flask culture Subheading 3. At the same time that this system was being developed, however, Tabachnick and Knudson 6 showed that four enzymes, i.
Third, the flow pattern is more consistent in the shaker flasks. The procedure essentially involves using a spectrophotometer for determining the activity in the sample. Another class of anti-apoptotic genes, the inhibitor of apoptosis iap genes, has also been identified. The author believes that errors as large as 40—50% on a linear scale are not unexpected. The medium in the culture should be fairly clear and cells should be somewhat refractive under the microscope Fig.