Transfection of insect cell lines using polyethylenimine.

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85-90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Baculovirus-based expression of an insect viral protein in 12 different insect cell lines.

The ability of 12 unique lepidopteran insect cell lines from Anticarsia gemmatalis, Heliothis virescens, Lymantria dispar (two lines), Mamestra brassica, Plutella xylostella, Spodoptera frugiperda (two lines), and Trichoplusia ni (three lines) to support production of a recombinant polydnavirus (PDV) protein (GiPDV 1.1) expressed using the Bac-to-Bac baculovirus expression system was examined. Polydnavirus gene GiPDV 1.1 was cloned into the pFastBac baculovirus vector under the control of the polyhedron promoter, followed by generation of recombinant bacmid-GiPDV 1.1 by site-specific transposition. The ability of each insect cell line to support recombinant PDV gene expression was estimated using reverse transcriptase-polymerase chain reaction and Western blot. Each insect cell line infected with recombinant bacmid-GiPDV 1.1 and tested in this study was capable of supporting and producing recombinant protein. Time course expression analysis showed that 72-96 h after transfection to be the optimal time for harvest of recombinant protein for each insect cell line.

Polydnavirus integration in lepidopteran host cells in vitro.

The long-term persistence of polydnavirus (PDV) DNA in infected lepidopteran cell cultures has suggested that at least some of the virus sequences become integrated permanently into the cell genome. In the current study, we provide supportive evidence of this event. Cloned libraries were prepared from two different Lymantria dispar (gypsy moth) cell lines that had been maintained in continuous culture for more than five years after infection with Glyptapanteles indiensis PDV (GiPDV). Junction clones containing both insect chromosomal and polydnaviral sequences were isolated. Precise integration junction sites were identified by sequence comparison of linear (integrated) and circular forms of the GiPDV genome segment F, from which viral sequences originated. Host chromosomal sequences at the site of integration varied between the two L. dispar cell lines but virus sequence junctions were identical and contained a 4-base pair CATG palindromic repeat. The GiPDV segment F does not encode any self-replication or self-insertion proteins, suggesting a host-derived mechanism is responsible for its in vitro integration. The chromosomal site of one junction clone contained sequences indicative of a new L. dispar retrotransposon, including a putative reverse transcriptase and integrase located upstream of the site of viral integration. A potential mechanism is proposed for the integration of PDV DNA in vitro. It remains to be seen if integration of the virus also occurs in the lepidopteran host in vivo.

Survival of Ranunculus repens L. (creeping buttercup) in an amphibious habitat.

The turlough form of Ranunculus repens is subjected to several months' complete inundation with hard groundwater. Experimental flooding to the level of the soil surface had no effect on turlough or ruderal populations relative to drained controls. Experimental submergence resulted in direct tissue death of the ruderal population but did not affect the turlough population relative to drained controls. There was no detectable difference in the proportion of aerenchyma in drained, flooded and submerged roots of plants from either population. The proportion of aerenchyma increased with root age in the ruderal population. Up to twice the proportion of aerenchyma occurred in the lower third of the root in the turlough population relative to the middle and upper thirds. Submergence in artificially hardened tap water increased the amount of tissue death in the ruderal population, whereas it appeared to enhance the growth of plants from the turlough population relative to that of plants submerged in tap water. Only the ruderal population demonstrated a depth accommodation response in submerged conditions. Root concentrations of ethanol-soluble carbohydrates were up to three times higher in a field- collected turlough population during winter and autumn months than those in a ruderal population. Low levels of ethanol-insoluble carbohydrates were present in the turlough population but were absent from the ruderal population. Starch concentrations fluctuated greatly in the turlough population and were generally higher than those in the ruderal population. These results, together with those from previous investigations, suggest that the turlough population survives prolonged submergence by maintaining low levels of submerged photosynthesis, which may circulate oxygen within the plant tissues, and by utilizing storage carbohydrates for maintenance respiration.

Physiological variation in populations of Ranunculus repens L. (creeping buttercup) from the temporary limestone lakes (turloughs) in the west of Ireland.

A dissected-leaved form of Ranunculus repens L. occurs in the temporary limestone lakes (turloughs) across the west of Ireland. Turloughs fill with groundwater for up to 8 months of the year. Under experimental conditions, these turlough populations demonstrated a higher rate of aerial and submerged photosynthesis than populations of the more typical broad-leaved ruderal form. The turlough populations also had higher rates of stomatal conductance and exhibited a higher stomatal index on the upper leaf surface and a lower index on the lower leaf surface than the ruderal populations. Neither population could utilize bicarbonate to any great extent, with rates of photosynthesis under submerged conditions being only 5 % of aerial rates. Respiration under submerged conditions was significantly higher in the turlough populations than in ruderal populations, and it is hypothesized that the more dissected leaf shape of the turlough population may have a thinner boundary layer and thus enhance gas exchange in submerged conditions.

Novel techniques to establish new insect cell lines.

The success of insect cell culture is demonstrated by reports of over 500 established cell lines. While established procedures that can be used for developing new cell lines exist, these usually require some fine-tuning for various tissue sources. This paper attempts to depict some of the variations that can be applied.

Effects of temperature on the susceptibility of insect cells to infection by baculoviruses.

Three insect cell lines were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure. Cell lines from Spodoptera frugiperda (IPLB-Sf21AE), Lymantria dispar (IPLB-LdEIta), and Heliothis virescens (IPLB-HvE6s) in 96-well tissue culture plates were each infected with dilutions of extra cellular virus suspensions of the Autographa californica nucleopolyhedrovirus (AcMNPV). In addition, the L. dispar and H. virescens cells were also infected with L. dispar nucleopolyhedrovirus, and Helicoverpa zea nucleopolyhedrovirus, respectively. Each cell/virus combination was incubated at three temperatures: 22, 27 and 32 degrees C and wells were scored for positive infection (presence of occlusion bodies in cell nuclei) at 2 to 4 day intervals for up to 4 weeks. The resulting data were analyzed by the Spearman-Kärber method, providing virus titers for each combination of virus, cell line, and temperature. The results were categorized by accuracy (assuming the highest titer achieved was the most accurate) and by rapidity of maximum titer. AcMNPV reached the highest titer in each line at 22 degrees C although equivalent titers were reached with both AcMNPV and HzSNPV in the HvE6a line at all three temperatures. This line actually reported about 100-fold less AcMNPV than the other two lines with the same virus sample. Alternatively, the Sf21AE and LdEIta lines reached 10-fold higher titers at the lowest temperature as compared with the higher temperatures, although also at a slower rate.

Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication.

Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM. These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies. The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production. These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.

Further characterization of the gonad-specific virus of corn earworm, Helicoverpa zea.

The gonad-specific virus (GSV) is a DNA virus infecting the reproductive tracts of adults of both sexes of the corn earworm, Helicoverpa zea, causing severe tissue deformities leading to sterility. Atypical occlusion bodies containing large concentrations of virions embedded in a granular matrix were seen in the lumen of the oviduct and the bursa copulatrix of infected females. The virus, transmitted by both sexes, was successfully propagated in vivo and in tissue culture. The GSV genome is about 225 kb in size, with no apparent similarity to the nucleopolyhedrovirus type species, AcMNPV, genomic DNA, as determined by Southern hybridization. PCR amplification of GSV genomic DNA with primers derived from the highly conserved polyhedra gene of several baculoviruses indicated no similarity. GSV at 10(-2) female equivalents (based on virus obtained from the bursa copulatrix and oviducts of one infected female) injected into a newly emerged female and mated to a normal male resulted in >95% agonadal progeny. However, at lower doses, some of the adult progeny looked normal but apparently carried a low level of the virus that could be responsible for sustenance of infection in a given colony, as well as in nature.

Transfection of Lymantria dispar insect cell lines.

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA.

Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R2, IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.

Comparison of cell line maintenance procedures on insect cells used for producing baculoviruses.

A gypsy moth cell line, IPLB-LdEIta, maintained under various conditions was tested for susceptibility to and productivity of two baculoviruses, the Autographa californica nucleopolyhedrovirus (AcMNPV) and Lymantria dispar nucleopolyhedrovirus (LdMNPV). The results suggest that cells maintained in serum-containing medium (modified TC100) were more susceptible (on the basis of titers in an endpoint assay) to LdMNPV than cells maintained in a serum-free medium (ExCell 400). Such a difference was not apparent with AcMNPV. Similarly, little difference existed in the proportion of cells containing occlusion bodies (OBs) a wk after inoculation with AcMNPV (i.e., the percent infected) in any LdEIta strains, although one combination of cells and medium (cells maintained in ExCell 400 but infected in TC100) showed a lower percent infection with LdMNPV. Even though the percentage of cells infected varied little, the number of OBs produced varied by 3 logs with AcMNPV and 11/2 logs with LdMNPV. In each case, cells normally grown in ExCell 400 and infected in the same medium produced the lowest number of OBs. However, productivity was improved when cells normally grown in ExCell 400 were infected in TC100. Even more interesting was that cells normally grown in TC100 produced more AcMNPV OBs when infected in ExCell 400 medium. This suggests that changing culture medium (regardless of the normal maintenance medium) can stimulate virus production. In addition to examining virus productivity in LdEIta cells in both serum-containing and serum-free media, I also tested a strain maintained at low temperature (17 degrees C) for over a yr. This maintenance protocol was not detrimental for LdMNPV productivity and was slightly stimulatory for production of AcMNPV.

Development of insect cell lines: virus susceptibility and applicability to prawn cell culture.

Insect cells have been successfully cultured in vitro as continuous cell lines for over 35 years. The media, culture methodology and conditions have been well resolved such that, for many insects, new cells lines can be routinely developed. Factors that are considered important for developing insect cell cultures are described as well as some of the history that led to the success. One of the major rationales for developing insect cell lines was for the study of insect viruses. This was particularly true for species of Lepidoptera from which over 900 viruses have been reported. Since many species of Lepidoptera are serious agricultural and forestry pests, effects have been made to utilize some of these pathogens as biological pesticides. Cell cultures are important in this endeavor since viruses require a living cell to reproduce. Of the known insect viruses, the most intensely studied have been the baculoviruses. In addition to their potential for controlling insect pests, they also have been used as expression vectors for producing recombinant proteins. Details of some of these experiments are described. Finally, experiences with insect cells are considered in relation to efforts to develop prawn cell cultures.

A new method for aversive Pavlovian conditioning of heart rate in rhesus monkeys.

Aversive Pavlovian conditioning is an important tool used to investigate neurobiological mechanisms underlying the acquisition and expression of fear. Most studies have used nonprimate species employing electrical shock as the unconditioned stimulus (US). Although important advances have been made in understanding the neural substrates of conditioned fear, the extent to which these findings apply to primates is unclear. Research in primates has not progressed because of the lack of a conditioning paradigm that does not use shock. Therefore, we developed a method that uses a US consisting of a loud noise coupled with a stream of compressed air aimed at the face to aversively condition heart rate response in rhesus monkeys. With this US, rhesus monkeys rapidly acquire a conditioned bradycardia. The availability of an easy, reliable, and efficient method of aversive conditioning that does not require electrical shock, will facilitate studies investigating neurobiological mechanisms underlying the acquisition and expression of fear in primates.

Transformation of gypsy moth (Lymantria dispar) cell lines by infection with Glyptapanteles indiensis polydnavirus.

Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells.

Immunoreactivities for calcium signaling components and neural-like properties of a Colorado potato beetle cell line.

Of the approximately 400 described insect cell lines, only three are derived from beetles (Coleoptera), and none are neural in origin. The present work was undertaken to characterize further a new cell line, designated IPLB-CPB2, derived from eggs of the Colorado potato beetle, Leptinotarsa decimlineata. Indirect immunofluorescent studies reported here indicate that these cells express neurofilament (Nf)-like immunoreactivities to antibodies directed against mammalian Nf-Medium (MW 150,000) and a heavily phosphorylated form of Nf-Heavy (MW 200,000; detected using the axonal monoclonal antibody SMI 31). This appears to be the first report of neurofilament-like immunoreactivity in an arthropod. Immunofluorescent analyses also indicate that IPLB-CPB2 cells express an antigenic epitope characteristic of the mammalian type 1 inositol trisphosphate (IP3) receptor, the ryanodine receptor (RyR) (also termed calcium-induced calcium release receptor channel, or CICR) and the sarco(endo)plasmic reticulum Ca2+ pump (SERCA). Patch-clamp recordings indicate that some IPLB-CPB2 cells are capable of producing spontaneous action potentials, while others may be photosensitive. Taken together, the findings reported here suggest that IPLB-CPB2 cells are of neural origin and that they express the major receptor channels and pumps known to be localized in the ER of the cell, which are required for receptor-mediated calcium signaling. Thus, the IPLB-CPB2 cell line may prove to be an excellent model system for studies of insect neurobiology and calcium-based signal transduction.

Development and characterization of insect cell lines.

CONCLUSIONS: With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the midgut, it may not be possible to obtain cell lines from these tissues from all insect species due to terminal differentiation and other factors. Also, researchers have desired cell lines from certain species, such as the honey bee, for which no success has been obtained. As in the early days of tissue culture, it is difficult to discern why negative results occur. However, as more is learned about the physiology and nutrition of various insects and tissues, we may get clues which will help solve these questions.The remaining chapters in this book will provide the reader with exciting uses for insect cell culture. As I mentioned earlier, the baculovirus expression vector system has provided a stimulus to the field of insect cell culture not seen previously.

Opiate systems in mother and infant primates coordinate intimate contact during reunion.

In humans, and non-human primates, reunion following a separation results in a positive emotional state, and an increase in affiliative behaviors. To examine the role of opiate systems, in mothers and infants in mediating reunion behavior, morphine and naltrexone were administered after a brief separation. Infants administered morphine (0.1 mg/kg IM) showed a significant reduction in clinging and girning, a vocalization emitted during close physical contact. Naltrexone (5 mg/kg IM) had opposite effects. When administered to mothers, again morphine decreased and naltrexone increased clinging. Morphine administered to mothers had a more transient behavioral effect which could not be accounted for by lower morphine blood levels. These results demonstrate that during reunion, the amount of intimate contact between a mother and her infant is regulated by the reciprocal activation of their opiate systems. This activation of opiate systems may reinforce the infant's need for attachment and the mother's role in care giving.