Patent Application: US-51876300-A

Abstract:
cell lines that are commonly used for protein expression are engineered to include genes that encode suppressors of apoptosis . insect cell lines expressing these sa genes are resistant to apoptosis or programmed cell death , and express certain recombinant proteins at increased levels . these cell lines also have increased resistance to many types of stress . because some of the sa proteins inhibit apoptosis in a wide spectrum of organisms , these genes may be inserted into other plant or animal cell lines for a variety of purposes involving resistance to apoptosis or resistance to stress .

Description:
the present invention satisfies the need in the art for improved cell lines resistant to apoptosis . the baculovirus acmnpv encodes a suppressor of apoptosis , p35 , which is expressed early during the infection cycle and is known to inhibit apoptosis triggered by acmnpv infection of sf21 cells . transient expression of p35 in sf21 insect cells is also known to prevent apoptosis . since the baculovirus already contains a p35 gene , it is not intuitive to assume that , by adding this gene to an insect cell line , it would confer the observed benefits to the infected cells . therefore , isolating a suppressor of apoptosis from a baculovirus and incorporating it into a cell line for this purpose is novel . sf9 cells were engineered to express p35 , a baculovirus suppressor of apoptosis . the resulting p35 - expressing cells were placed under selection using an inducer of apoptosis , such as actinomycin d . cell lines cloned by this procedure expressed p35 , were more highly resistant to nutrient stress , and yielded secreted recombinant proteins from baculovirus infections at levels similar to that achieved by tn5b1 - 4 cells , currently one of the best cell lines available for recombinant protein production from baculovirus - infected cells . to generate cell lines resistant to induction of apoptosis by stressful culture conditions , and to examine the effects of constitutive cellular p35 expression on protein production from baculovirus expression vectors , cell lines expressing acmnpv p35 or an epitope - tagged p35 fusion protein were generated . cell lines expressing p35 were first selected using a neomycin resistance gene and g418 , then selected again in the presence of actinomycin d , an inducer of apoptosis in sf9 cells . several clonal isolates were generated and examined for a ) resistance to actinomycin d induced apoptosis and nutrient deprivation , b ) growth in various media , and c ) baculovirus expression of intracellular and secreted proteins . when compared with wild type sf9 cells , two p35 expressing cell lines showed increased resistance to actinomycin d induced apoptosis , and a marked resistance to nutrient deprivation . when these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein ( secreted alkaline phosphatase , seap ), expression of the glycoprotein from these cells exceeded expression from wild type sf9 cells , and was comparable to expression levels obtained from tn5b1 - 4 cells . proteins expressed in these cells may also be of higher quality , as protein processing may be , on average , more uniform or more complete than in the parental cells . the acmnpv p35 gene ( seq . id . no . 1 ; ayres , m . d . et al ., 1994 ) was amplified from the acmnpv genome and subcloned into an insect cell expression plasmid ( p166brnx ) which contains an optimized baculovirus early promoter ( blissard , g . w . and rohrmann , g . f ., 1991 , monsma , s . a ., oomens , a . g . p . and blissard , g . w ., 1996 ). although p35 may have more powerful inhibitory effects than other sas , other suppressors of apoptosis , which include bombyx mori p35 and inhibitors of apoptosis proteins ( iaps ) from other baculoviruses , can be used similarly in the development of novel cell lines . each of these sas is derived from a baculovirus , and is not a part of the genome of the parental cell line . two p35 insect cell expression plasmid constructs were generated . in one construct , the wild type acmnpv p35 gene was cloned in its native form ( amino acids 1 - 299 ). a second construct encoded the full length p35 orf with an acv5 epitope tag ( monsma , s . a . and blissard , g . w ., 1995 ) fused to the c - terminus of p35 ( fig1 ). the p35 amino acid sequence is shown in seq . id . no . 1 and seq . id . no . 2 ( ayres , m . d . et al ., 1994 ). a plasmid ( p166brnx - acv5 ) for insect cell expression was constructed from the opmnpv gp64 promoter region , a multiple cloning site , sequences encoding the acv5 epitope , and polya addition sequences from the opmnpv gp64 gene ( fig1 ). the acmnpv p35 orf was pcr amplified and cloned into the ecori and xbai sites . to construct this plasmid , a complementary oligonucleotide pair ( seq . id . no . 3 and seq . id . no . 4 ) encoding a 5 ′ xba i site , the acv5 epitope ( swkdasgws ; monsma , s . a . and blissard , g . w . 1995 ), two stop codons , and an xbai compatible cohesive end at the 3 ′ end was synthesized and cloned into plasmid vector p166brnx digested with xbai . the resulting plasmid , p166brnx - acv5 , contains a 166 nucleotide opmnpv gp64 early promoter region ( blissard , g . w . and rohrmann , g . f ., 1991 ) followed by a multiple cloning site containing bamhi , ecori , eagi , noti , and xbai sites , plus a polyadenylation signal from the opmnpv gp64 gene . this plasmid was used to create the constructs below . the p35 orf was cloned in frame with the acv5 epitope such that the expressed p35 protein contains a 2 amino acid linker and a 9 amino acid acv5 epitope at the c - terminus ( fig1 ). the acmnpv p35 orf ( with no stop codon ) was amplified by pcr from acmnpv genomic dna using two primers , p35upecori ( seq . id . no . 5 , a primer that contained the sequences from the 5 ′ end of the p35 orf plus an ecori site at the 5 ′ end ) and p35lowxbai - no stop ( seq . id . no . 6 , a primer that contains sequences from the 3 ′ end of the p35 orf , but no stop codon , and a 3 ′ xbai site ). the pcr product was cloned into ecori / xbai digested plasmid p166brnx - acv5 to obtain plasmid p166 - p35 - acv5 . plasmid p166 - p35 - acv5 contains the p35 orf fused to a c - terminal acv5 epitope tag , under the control of the opmnpv gp64 early promoter ( fig1 ). construct p166 - p35 includes a stop codon upstream of the acv5 epitope and expresses a native p35 protein ( fig1 ). the p35 gene was amplified using primers p35upecori ( seq . id . no . 5 ) and p35lowxbai - stop ( seq . id . no . 7 ) using the same strategy described above , except that the p35 stop codon was included in the 3 ′ oligonucleotide and pcr product ( fig1 ). the resulting plasmid , p166 - p35 , expresses wild type p35 . all of the constructs were confirmed by dna sequencing . each p35 expression plasmid was cotransfected into sf9 insect cells with a plasmid ( pie1 - neo ) expressing neomycin phosphotransferase ( npt ) and cells were selected using geneticin disulfate ( g418 ). plasmid pie1 - neo contains a bacterial npt gene under the control of an acmnpv ie1 promoter ( monsma , s . a ., oomens , a . g . p . and blissard , g . w ., 1996 ). cells were also cotransfected with plasmid p166 - egfp , which contains an enhanced green fluorescent protein gene under the transcriptional control of the opmnpv gp64 early promoter ( chang , m . j ., kuzio , j . and blissard , g . w ., 1999 ). egfp expression was used as a convenient visible marker for transgene expression in stably transfected cell lines . other parental cell lines available for production of stably transfected cell lines include iplb - sf21 , bti - tn5b1 - 4 , bti - mg - 1 , tn368 , ld652y , and bti - eaa , any cell lines derived from the cell lines listed here , as well as any cell line susceptible to baculovirus infection . those skilled in the art would appreciate that , in order to meet their unique expression needs , this method is applicable to cell lines not specifically listed . examples of some of these cell lines are found in granados , r . r . and hashimoto , y ., 1989 . sf9 cells were transfected using the capo 4 technique being known in the art , and herein incorporated by reference ( blissard , g . w . and rohrmann , g . f ., 1991 ). to generate cells expressing the epitope tagged p35 protein , sf9 cells ( 2 × 10 6 cells ) were transfected with plasmids pie1 - neo ( 1 μg ), p166 - p35 - acv5 ( 5 μg ), and p166 - egfp ( 1 μg ). to generate cells expressing the native p35 protein , sf9 cells were similarly transfected with plasmids pie1 - neo ( 1 μg ), p166 - p35 ( 5 μg ), and p166egfp ( 1 μg ). at 48 hours post transfection , g418 was added to the medium to a final concentration of 1 mg / ml and cells were incubated in g418 containing medium for three to four weeks . under these conditions , only sf9 cells that were stably transfected survived . cells selected in g418 were also screened visually for egfp fluorescence . untransfected sf9 cells were used as a negative control . in order to select cells expressing higher levels of functional p35 , the surviving transfected cells were placed in medium containing 0 . 1 μg / ml actinomycin d for 1 hour . another example of an inducer of apoptosis that could be used to select cells resistant to apoptosis is uv irradiation . in mammalian cells , inducers of apoptosis such as tumor necrosis factor ( tnf ) could also be used . the selection using an inducer of apoptosis could alternatively be performed without cotransfection with a selectable marker such as an antibiotic resistance gene . at the end of the one hour incubation in actinomycin d , the medium was replaced with fresh tnmfh medium ( containing no actinomycin d ) and cells were allowed to grow for three days . after three days , 85 - 90 % of the cells treated with actinomycin d - containing medium died . medium was replaced after 3 days and surviving single cells formed small colonies which were subsequently propagated and used to clone individual cell lines by limiting dilution . for limiting dilution cloning , cells were diluted into 96 well plates such that each well received only one cell on average . each well was subsequently monitored to ensure that only a single colony was present . monoclonal cell lines were selected and propagated . cell lines expressing the native p35 protein were named sf9 p35 , and lines expressing the epitope tagged p35 protein were named sf9 p35acv5 . five stable cell lines expressing p35 were selected and named sf9 p35 − 1 , sf9 p35 − 2 , sf9 p35 − 3 , sf9 p35 − 4 , and sf9 p35 − 5 . three cloned cell lines expressing the epitope tagged p35 protein were selected and named sf9 p35acv5 − 1 , sf9 p35acv5 − 2 , and sf9 p35acv5 − 3 . a sample of two new cell lines , designated bti - sf9 - p35acv5 - 1 and bti - sf9 - p35acv5 - 3 , was deposited on feb . 23 , 2001 with the american type culture collection , at 10801 university blvd ., manassas , va . 20110 - 2209 , under accession no . pta - 3099 and no . pta - 3100 . growth rates of selected cell lines were determined by plating 1 × 10 6 − 2 × 10 6 cells in tnmfh medium supplemented with 10 % fetal bovine serum in t - 25 flasks , and monitoring cell growth at 24 hours intervals by a technique being known in the art , and herein incorporated by reference ( wang , p ., granados , r . r . and shuler , m . l ., 1992 ). growth curves were generated for cell lines sf9 p35 − 1 , sf9 p35 − 3 , sf9 p35 − 4 and sf9 p35 − 5 , sf9 p35acv5 − 1 , sf9 p35acv5 − 2 , and sf9 p35acv5 − 3 . to determine whether the growth rates of cell lines expressing p35 were affected by p35 expression or stable transfection , the growth curves were compared to the growth rate of sf9 cells ( table 1 ). under these conditions , the average doubling time for unmodified sf9 cells was approximately 26 hours . doubling times for stably transformed cell lines ranged from 23 to 39 hours , with most lines showing similar doubling times to sf9 cells . only cell lines sf9 p35 − 3 and sf9 p35 − 4 showed growth rates that were significantly extended in comparison to the parental sf9 cell line . the novel cell lines may also preferably contain a recombinant dna for expression of a recombinant protein . for example , two recombinant baculovirus constructs that express well characterized proteins were used to compare recombinant protein production in the cells stably transfected with p35 with production from standard insect cell lines . virus racmnpv - seap encodes a truncated human placental alkaline phosphatase gene under the control of the acmnpv polyhedrin promoter ( davis , t . r ., trotter , k . m ., granados , r . r . and wood , h . a ., 1992 ). this virus expresses secreted alkaline phosphatase ( seap ), a glycoprotein that is conveniently monitored by measuring seap activity from cell culture supernatants ( davis , t . r . et al ., 1993 ). a second virus , acmnpv - 246 ( wickham , t . j ., davis , t ., granados , r . r ., shuler , m . l . and wood , h . a ., 1992 ), contains an e . coli lacz gene under the control of the acmnpv polyhedrin promoter . beta - galactosidase assays of infected cell lysates were used to measure the synthesis of this intracellular protein . to examine levels of seap expression from sf9 or stably transfected cells expressing p35 in serum - containing medium , cells were plated in 24 well plates . for sf9 cells and stably transfected cells expressing p35 , 3 × 10 5 cells / well were plated in each well of 24 well plates . due to their larger size , tn5b1 - 4 ( highfive ™) cells were plated at a density of 1 × 10 5 cells / well . cells were infected with racmnpv - seap at a multiplicity of infection ( moi ) of 10 for 1 hour , then virus was removed and cells were placed in fresh medium . supernatants were collected from infected cells at 2 , 3 , 4 , 5 , 6 , 7 , 8 , or 9 days post infection ( p . i .). each time point in fig2 represents seap accumulation from initiation of infection through the indicated time . for each time point , three separate replicate wells were infected and separate supernatants collected . seap activity was determined by a technique being known in the art , and herein incorporated by reference ( davis , t . r ., trotter , k . m ., granados , r . r . and wood , h . a ., 1992 ). tn5b1 - 4 cells are substantially larger than sf9 cells . therefore , to accurately compare expression of seap from stably transfected sf9 cells with that from tn5b1 - 4 cells , seap activity was calculated on both a “ per cell ” and a “ per biomass ” basis and examined as : 1 ) international units ( iu )/ cell and 2 ) iu / mg cell protein . the total biomass of the sf9 and tn5bi - 4 cells were compared using total protein content per cell to indicate biomass . as estimated from bradford protein assays , tn5b1 - 4 cells contained approximately 0 . 353 mg protein per 10 6 cells , whereas sf9 p35acv5 − 1 cells ( a representative cell line derived from sf9 cells ) contained approximately 0 . 208 mg protein per 10 6 cells . thus , based on protein content , the biomass of an average tn5b1 - 4 cell is almost 1 . 5 times that of an average sf9 cell . levels of seap were determined and are presented as international units ( iu ) seap per mg cell protein ( iu / mg cell protein ) in fig2 . as expected , seap expression from tn5bi - 4 cells exceeded that from sf9 cells , at most times by approximately 2 - 3 fold when compared on a biomass basis ( fig2 , 3 - 9 days post infection ). seap expression levels from all p35 - expressing cell lines were higher than those from the parental sf9 cells , and were generally comparable to seap levels from tn5bi - 4 cells , with the exception of measurements at 2 - 3 days post infection when seap levels in tn5bi - 4 cells exceeded all others ( fig2 ). seap accumulated to very high levels in one of the p35 - expressing lines , sf9 p35acv5 − 1 . seap levels in line sf9 p35acv5 − 1 were similar to those from tn5b1 - 4 cells at 3 - 4 days post infection , but continued to accumulate to significantly higher levels (≧ 2 - 3 fold above sf9 cell expression ) at 5 - 6 days post infection . by 7 - 9 days post infection , seap levels from the sf9 p35acv5 − 1 line exceeded that from parental sf9 cells by approximately 4 fold and were approximately 2 fold more than seap levels from tn5b1 - 4 cells . high level protein production in sf9 p35acv5 − 1 cells appears to result from a prolonged infection cycle compared with the infection cycle of acmnpv infected sf9 cells . in typical baculovirus expression vector infection of sf9 or tn5b1 - 4 cells , foreign protein accumulation plateaus after approximately 120 hours post infection . in contrast , secreted proteins continued to accumulate until 216 hours post infection in both sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells sf9 or stably transfected cells expressing p35 were plated at 3 × 10 5 cells / well and tn5b1 - 4 cells were plated at 1 × 10 5 cells / well in 24 well plates to measure levels of b - galactosidase . cells were infected with virus acmnpv - 246 ( which contains an e . coli lacz gene and expresses the b - galactosidase reporter protein ) at an moi of 10 as described above . cells were collected at 2 , 3 , 4 , 5 , 6 , 7 , 8 , or 9 days post infection for production of lysates ( fig3 ). analysis of b - galactosidase activity was performed essentially by a technique being known in the art , and herein incorporated by reference ( wang , p ., granados , r . r . and shuler , m . l ., 1992 ). lysates from each sample were prepared by placing cells in 100 μl pbs , then freezing and thawing the cells three times ( 10 min at − 70 ° c ., 10 min at 37 ° c .) followed by centrifugation for 5 minutes at 12 , 000 × g to remove debris . supernatants were decanted and 20 μl was used for each b - galactosidase reaction . each b - galactosidase reaction was incubated at 28 ° c . and b - galactosidase activity was monitored by readings at od420 nm and compared to a standard curve . international units ( iu ) of b - galactosidase activity were calculated as described earlier ( yu , z ., podgwaite , j . d . and wood , h . a ., 1992 ). for each time point in fig3 , three infections were performed . data collected at each time point represents b - galactosidase accumulation from initiation of infection through the indicated time . because the cell types compared in this study differ in size and volume , expression levels of b - galactosidase were calculated as 1 ) international units ( iu )/ cell and 2 ) iu / mg cell protein . cell lines expressing acmnpv p35 or tagged p35 were compared with sf9 and tn5b1 - 4 cell lines . generally , in infected p35 - expressing cell lines , b - galactosidase levels were slightly higher than those observed in the infected control sf9 cells ( fig3 ). one stably transfected p35 line , sf9 p35acv5 − 1 , exhibited b - galactosidase expression levels that consistently exceeded those from sf9 cells , with increased expression in this case ranging from approximately 2 . 3 - 2 . 7 fold from 4 - 8 days post infection . thus , one p35 - expressing cell line was superior to sf9 cells for expression of b - galactosidase . however , expression from line sf9 p35acv5 − 1 did not exceed that in tn5b1 - 4 cells under these conditions , as b - galactosidase expression in tn5b1 - 4 cells was consistently higher than both sf9 and the best stably transfected p35 - expressing line ( fig3 , tn5b1 - 4 vs . sf9 p35acv5 − 1 ). serum - free media formulations are routinely used for cell propagation and protein production from recombinant baculoviruses . for comparisons of sf9 and p35 expressing cells in serum - free medium , sf9 , tn5b1 - 4 , and stably transfected p35 lines sf9 p35acv5 − 1 and sf9 p35acv5 − 3 were initially grown in tnmfh medium supplemented with 10 % fetal bovine serum . these cells were subsequently adapted to serum - free medium ( sf900 - ii ; life technologies , inc ., rockville , md .). after adaptation to serum - free medium , these cells were passaged approximately eight times . then , cells were infected with virus racmnpv - seap and seap assays performed on cell culture supernatants as described above . supernatants were collected at 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 days post infection and seap levels determined . when seap activity in serum free medium was examined , it was observed that , from 3 - 9 days post infection , accumulated seap levels in tn5bi - 4 cells were significantly higher ( 1 . 54 - 2 . 6 fold ) than in sf9 cells ( fig4 ). also , from 5 - 9 days post infection , seap expression from stably transfected p35 - expressing cells was significantly higher ( 1 . 7 - 2 . 2 fold ) than in sf9 cells ( fig4 ). generally , seap expression levels from stably transfected p35 cells was similar to that from tn5b1 - 4 cells , although one cell line ( sf9 p35acv5 − 3 ) appeared to have slightly higher levels than that from tn5bi - 4 cells from 7 - 9 days post infection . increasing numbers of cells ( 0 . 5 × 10 6 , 1 × 10 6 , and 2 × 10 6 cells ) from each cell line were examined on western blots and relative p35 levels were quantified by fluorescence imaging . sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells were collected and washed twice with pbs . proteins were denatured by heating to 100 ° c . for 5 minutes in laemmli buffer and electrophoresed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) gel . proteins were transferred to immobilon - p membrane ( millipore corp ., bedford , mass .) and the epitope - tagged p35 protein was detected using monoclonal antibody acv5 ( 1 : 500 dilution ) as a primary antibody , and a goat anti - mouse igg alkaline phosphatase conjugate as a secondary antibody ( 1 : 10 , 000 dilution ). for semi - quantitative comparisons , an ecf substrate , diethanolamine ( amersham pharmacia biotech , piscataway , n . j . ), was used for protein immunodetection on a storm laser scanning system ( molecular dynamics , inc .). fig5 shows the relative p35 acv5 expression levels for p35 acv5 − 1 and p35 acv5 − 3 cells . the 1 × column shows the relative expression levels from 0 . 5 × 10 6 cells , while the 4 × column shows the relative expression levels from 2 . 0 × 10 6 cells . p35 fusion proteins were identified by incubation with mab acv5 and proteins were quantified by enhanced chemifluorescence . from these comparisons , it was estimated that the acv5 - tagged p35 protein from line sf9 p35acv5 − 3 was present at levels approximately 1 . 5 - 1 . 7 × above that detected from line sf9 p35acv5 − 1 . thus , the levels of p35 detected in the two cell lines do not differ substantially . southern blot hybridization analysis was used to determine p35 gene copy number in stably transfected cell lines . a pcr amplified 908 bp dna fragment containing the acmnpv p35 gene open reading frame was labeled with either digoxigenin ( dig ) using random primers ( dig high prime labeling and detection starter kit 1 , boehringer mannheim company ) or with 32 p - datp ( decaprime 11 random priming kit , ambion , inc ., austin , tex . ), and used as a probe for high stringency hybridization analysis . for southern blots , 20 μg dna from each cell line ( sf9 p35acv5 − 1 or sf9 p35acv5 − 3 ) was digested with ecori and xbai , and electrophoresed and blotted onto positive charged nylon membrane ( micron separations inc .). either 0 . 1 , 0 . 3 , 0 . 5 , 0 . 7 or 1 . 0 μg p166 - p35 - acv5 plasmid dna ( digested with ecori and xbai ) was mixed with 20 μg sf9 cell dna ( also digested with ecori and xbai ), electrophoresed and blotted to generate a standard curve for quantitative analysis . increasing quantities of plasmid dnas in each lane simulated increasing copy numbers of the p35 gene in the sf9 genome . hybridization data from these experiments was used to generate a standard curve for gene copy number . two separate experiments were performed to compare a standard curve of p35 dna to p35 dna detected in lines sf9 p35acv5 − 1 and sf9 p35 acv5 − 3 . the haploid sf9 cell genome was estimated as approximately 5 × 10 8 base pairs , based on the approximated size of the bombyx mori genome ( rasch , e . m ., 1974 , gage , l . p ., 1974 ). estimates for gene copy number per haploid genome in sf9 p35acv5 − 1 and sf9 p35acv5 − 3 were derived from comparisons of hybridization signal strength between these cell lines and the standard curve reconstructed from plasmid dna . data were adjusted to the estimated size of the sf9 genome . the data suggested that line sf9 p35acv5 − 1 contains approximately 12 copies of the p35 gene per haploid genome , while line sf9 p35acv5 − 3 contains approximately 2 copies of the p35 gene per haploid genome . protein expression levels for these two cell lines do not correlate well with the relative number of copies of p35 in the genome . a possible explanation for this fact is that expression levels may be more dramatically affected by the site of integration rather than by the number of integration events . resistance of the cell lines to actinomycin d and nutrient stress the cells were examined for resistance to induction of apoptosis by actinomycin d , as well as survival under conditions of nutrient deprivation . sf9 cells or stably transfected lines sf9 p35acv5 − 1 and sf9 p35acv5 − 3 were incubated in medium containing 0 . 1 μg / ml actinomycin d for 1 hour , then placed in tnmfh medium . after three days , all sf9 cells died , while both sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells survived . thus , both p35 - expressing cell lines exhibited resistance to actinomycin d . increasing doses of actinomycin d were tested to further examine the degree of resistance to this inducer of apoptosis . sf9 or stably transfected cells expressing p35 were incubated in a range of concentrations of actinomycin d ( 0 . 01 to 0 . 5 μg / ml ) for one hour , then placed in tnmfh and scored for cell survival after 1 - 4 days ( table 2 ). apoptosis was induced in wild type sf9 cells at actinomycin d concentrations between 0 . 05 μg / ml and 0 . 1 μg / ml ( and higher ) ( table 2a ), whereas both p35 - expressing cell lines ( sf9 p35acv5 and sf9 p35acv5 − 3 ) were sensitive to actinomycin d only at higher concentrations between 0 . 25 μg / ml and 0 . 5 μg / ml ( table 2b and 2c ). resistance to actinomycin d as an inducer of apoptosis appears to be ≧ 2 fold , since survival of sf9 cells was high ( approximately 80 - 95 %) at 0 . 075 μg / ml actinomycin d and was reduced to ≦ 5 % at 0 . 1 μg / ml ( table 2a ). in contrast , survival of both stably transfected lines was 100 % at 0 . 25 μg / ml actinomycin d , and both were reduced to ≦ 10 % at 0 . 5 μg / ml ( table 2b and 2c ). to further examine resistance to apoptosis by p35 expressing cells , sf9 , sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells were plated at 2 × 10 6 cells / well in 6 - well plates , then exposed to actinomycin d ( 0 . 1 μg / ml ) for one hour , and incubated in fresh tnmfh medium at 27 ° c . for 24 hours . cells were harvested and washed twice with pbs . lysates were prepared by incubating cells in 200 μl lysis buffer ( 10 mm tris ph 7 . 5 , 25 mm edta , 0 . 2 % triton x - 100 ) for one hour at room temperature . the lysate was extracted once with phenol , once with phenol : chloroform ( 1 : 1 ), twice with chloroform , and then precipitated in two volumes of ethanol . cellular dna was resuspended in 30 μl water containing rnase a ( 50 μg / ml ). 10 μl of dna from each cell treatment was electrophoresed on a 1 . 2 % agarose gel in tbe buffer ( not shown ). examination of the dna on ethidium bromide stained gels indicated that treatment of sf9 cells with actinomycin d resulted in a dna laddering effect typical of apoptosis . degradation of the dna into dna ladders ( compared to an untreated control ) was apparent in the electrophoresed dna from sf9 cells . in contrast , the treated dna extracted from both sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells showed little or no laddering or degradation . thus , induction of apoptosis was not observed in sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells after exposure to 0 . 1 μg / ml actinomycin d . these results are consistent with the cell survival studies shown in table 2 . since lines sf9 p35acv5 − 1 and sf9 p35acv5 − 3 were resistant to induction of apoptosis , these lines were also examined for resistance to nutrient stress by culturing cells in phosphate buffered saline ( with no nutrients added ) for extended periods . sf9 , sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells were plated in tnmfh medium . after a 2 - hour attachment and equilibration period , tnmfh medium was removed and replaced with pbs ( ph 7 . 1 ) and incubated at 27 ° c . for 9 days . cells were scored for viability at 24 hours intervals . the percentage of cells surviving at daily intervals and a comparison of cell survival are shown in fig6 . for example , when cells were examined at 20 hours post infection , sf9 p35acv5 − 3 cells were rounded with a large nucleus , dense cytoplasm , and little or no granularity of the cells . essentially , these cells appeared healthy and intact . in contrast , sf9 cells similarly incubated in pbs and examined at 20 hours post infection were mostly granular and shriveled in appearance , indicative of extensive cell lysis . after 24 hours in pbs , viability of sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells was approximately 85 % and 55 % respectively . in contrast , viability of sf9 cells was less than 5 % at the same time point . line sf9 p35acv5 − 3 exhibited the greatest degree of resistance to nutrient stress as viability of sf9 p35acv5 − 3 cells remained above 40 % after 4 days in pbs . lines sf9 p35acv5 − 1 and sf9 p35 − 3 also showed much greater resistance to nutrient stress than sf9 cells , but in these lines cell viability dropped from ≧ 55 % at 1 day , to around 15 - 20 % after 3 days in pbs . no surviving sf9 cells were observed after 4 days in pbs . notably , some cells stably expressing p35 ( sf9 p35acv5 − 1 and sf9 p35acv5 − 3 ) remained viable through 7 - 8 days in pbs . thus , the stably transfected cell lines show an extraordinary resistance to nutrient deprivation when compared with unmodified sf9 cells . to examine the potential long - term effects of nutrient deprivation on sf9 p35acv5 − 1 and sf9 p35acv5 − 3 cells , these cells were placed in pbs buffer for 5 days as described above , then returned to tnmfh medium . in both cases , cells recovered and grew normally . when infected with the racmnpv - seap virus , expression levels of seap in the supernatant were similar to those from the same cell lines which had not been exposed to nutrient stress ( not shown ). thus , exposure to extreme nutrient stress did not appear to affect the capacity of these cells to express recombinant proteins at high levels . foreign gene expression in insect cells has proven to be a tool of major importance in research , as well as industrial scale production of recombinant proteins . sf9 cells are well suited for large scale culture applications , as they are known to express reasonably high levels of recombinant proteins and they adapt readily both to suspension culture and serum - free media preparations . however , sf9 cells are somewhat fastidious , since they do not readily tolerate nutrient stress or high - density growth . the invention demonstrates that by stably expressing a viral suppressor of apoptosis , such as the baculovirus p35 protein , in an insect cell line , such as sf9 , novel cell lines are generated and these cells are resistant to the induction of apoptosis , resistant to nutrient stress , and support substantially higher levels of protein expression than the parental cell line . the stable transfection of viral genes capable of suppressing apoptosis into a pre - existing cell line creates more hardy cells . the novel cell lines overcome the disadvantages of their parental cell line , sf9 . these cells express levels of recombinant proteins ( secreted ) at levels higher than that from the parental cell line ( sf9 ) and comparable to the most highly productive cell line available ( tn5b1 - 4 ). it is not entirely clear how expression of p35 from the cell line results in these phenomena , since p35 is normally expressed from acmnpv early in infection . it is possible that the accumulation of p35 in the cell prior to infection permits the infection to persist in the cells for longer periods or facilitates the maintenance of a more healthy cellular physiological state throughout infection . the presence of p35 in the cell before infection provides substantial benefits , including the expression of foreign genes . characterized cell lines derived from sf9 cells and expressing the acmnpv p35 gene are easily adapted to serum - free medium and grow readily in suspension cultures . engineered suppressor of apoptosis genes , such as the acmnpv p35 gene , as well as plasmids containing the suppressor of apoptosis gene ( s ) and antibiotic resistance genes , are useful for engineering other cell lines . as an example , viral sa proteins could be engineered for expression in mammalian cells such as cos cells or nih3t3 cells , for improved culture characteristics . the incorporation of viral sa proteins may affect production of valuable proteins in these cells or make them susceptible to additional pathogens . the replication of certain viruses in certain mammalian cell lines may be limited by the induction of apoptosis , which prevents productive infection and virus replication . by incorporating viral sa proteins into these cells , they may become useful for new applications , including protein expression in mammalian cells or the propagation or study of other viruses ( for example , retroviral vectors ). the use of suppressors of apoptosis such as p35 improves cell lines for protein expression with baculovirus expression vectors . the p35 - expressing cells have good market potential for industrial and / or pharmaceutical applications in which scaleup production of insect cells ( prior to infection ) is necessary , and high - level protein production is essential . in addition , sf9 or other cell lines can be engineered with p35 or other suppressors of apoptosis . the technology is easy to apply to existing expression systems . it requires only the use of a different cell line for expression of foreign proteins . the invention permits increased flexibility in handling and scale - up of cell lines for many applications . accordingly , it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention . reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims , which themselves recite those features regarded as essential to the invention . the following references are cited in the application and hereby incorporated by reference in their entirety . ayres , m . d ., howard , s . c ., kuzio , j ., lopez - 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