Patent Application: US-201414773252-A

Abstract:
provided is a gene expression system , suitable for expression in an insect , comprising an insect muscle actin pro - moter operably linked to a marker gene , which overcomes or ameliorates one or more of : cost of rearing ; amount of handling ; errors in identification due to human error or loss of marker by the insect ; and health concerns related to the effects of marker powders on workers in mass rearing facilities .

Description:
systems of the present invention may be used in order to express any suitable marker protein . for the sake of convenience , such marker proteins will frequently be referred to as fluorescent proteins herein , but it will be appreciated that any such reference includes reference to all suitable marker proteins , unless otherwise apparent from the context . expression of a fluorescent protein is desirable for several applications including ( i ) identification of transgenics in the process of transformation , ( ii ) identifying the presence of the transgene in laboratory strains and experiments , ( iii ) identifying the presence of the transgene in released insects and their descendants , e . g . following trapping . the expression should be strong so as to be readily scored , e . g . against background , or in older specimens ( e . g . which have been dead for some time before scoring ). the expression should ideally be in most , or all , body parts and developmental stages , so that presence can be determined in all or most life cycle stages and in incomplete or damaged specimens . yet expression , especially strong expression , may impose undesirable fitness penalties , such as those driven by cytoplasmic actin promoters , as explained above , or weak expression . accordingly , it is desirable to restrict expression to a single tissue . this should improve fitness unless the tissue happens to be particularly sensitive to expression . muscle fits the above criteria . muscle promoters make strong candidates for such expression given that muscle tissue is ample in any organism both spatially and temporally . furthermore , it is possible to systematically identify , by homology , highly expressed muscle - specific proteins , which are the structural proteins of muscles . we have identified such a muscle promoter and shown that it does indeed give very strong , clear expression of a fluorescent reporter in two tephritid fruit flies , and in moths , plutella xylostella and pink bollworm . moths in general , and these species in particular , are a preferred target insect in which the system is expressed . it is preferred to distinguish between muscle actin and non - muscle , or cytoplasmic actin , which is found in essentially every cell and which , therefore , tends to have much broader expression in terms of tissue and cell types . a further benefit of tissue - specific expression , rather than constitutive expression , is that expression in different tissues can , in principle , be scored independently . moreover , it is preferred to distinguish between muscle actin and indirect flight muscle actin ( allen m l , christensen b m 2004 ). expression of indirect flight muscle actin is substantially restricted to the indirect flight muscle . consequently , it is expressed in fewer body regions , essentially just the thorax , and in fewer developmental stages , being late larvae , pupae and possibly through into adults , than ‘ conventional ’ muscle actins . as noted above , restricted spatial expression markers make it ineffective for field monitoring purposes , as trapped insects may lose significant part of their bodies in a trap , resulting in misidentification and recording of the caught insects in a control programme . accordingly , in some embodiments , the indirect flight muscle actin is excluded , especially act88f from drosophila . there are various muscle actin promoters in some insects , so the invention preferably provides those that express across at least two , and preferably 3 , or even all , life cycle stages , which is clearly an advantage for detection , for instance . thus , any two of egg , larval , pupal or adult expression is preferred , and preferably consecutive stages . in addition to , or in combination with , any of theses , it is also preferred that that expression is across at least two and preferably all body segments of an insect , being the head , thorax and abdomen . it is particularly preferred that the expression is seen in all , or at least the abdomen and / or thorax , body sections in adults . surprisingly , despite widespread and strong expression of the fluorescent protein conferred by the muscle actin promoter , we have found that no fitness disadvantage was conferred on the transgenic insect . thus , the gene expression system of the present application obviates the need for the use of dyes or powders and alleviates the disadvantages associated with these methods , including those of adverse health effects on the handlers and the possibilities of human error . as such , it is preferred that expression of the marker gene , e . g . a fluorescent protein , under the control of the muscle actin promoter is sufficiently strong that the transgenic individuals are easily recognizable . this is particularly the case if they are recognisable by the naked eye . ideally , this will be at all , or two or 3 , of the larval , pupal and , in some cases , adult stages . it is also preferred that the expression , preferably strong expression , of the marker gene , such as fluorescent protein , confers little or no fitness disadvantage , preferably such that little or no the effects of the transgene are observed in any of the transgenic strains . actin is a globular multi - functional protein that forms microfilaments . it is present in all eukaryotic cells at high concentrations . in muscle fibres , actin comprises 20 % of total cellular protein by weight . actin has been one of the most highly conserved proteins throughout evolution , because it interacts with a large number of other proteins . it has 80 . 2 % sequence conservation at the nucleotide level between homo sapiens and saccharomyces cerevisiae , and 95 % conservation of the primary structure of the protein product . although most yeasts have only a single actin gene , higher eukaryotes , in general , express several isoforms of actin encoded by a family of related genes . mammals have at least six actin isoforms coded by separate genes , which are divided into three classes , alpha , beta and gamma , according to their isoelectric points . in general , alpha actins are found in muscle , whereas beta and gamma isoforms are prominent in non - muscle cells . although the amino acid sequences and in vitro properties of the isoforms are highly similar , these isoforms cannot completely substitute for one another in vivo ( perrin and ervasti , 2010 ). considering the high expression of muscle actin in eukaryotic organisms , indicative of strong promoter sequences , and the conserved nature of the genes involved , we decided to identify and utilise these promoters as potential transformation markers with reliable field properties ; i . e . sustainable fluorescent expression in dead tissue . to this end , the muscle actin promoter from the mexican fruit fly , anastrepha ludens , was identified and isolated as described in the example below . it should be noted that there will often be more than one muscle actin gene in an insect genome . a person skilled in the art would recognise that any variant of the muscle actin promoter may be used with the gene expression system described herein . the gene expression system may also comprise further genes ( i . e . polynucleotide sequences for expression , e . g . of a transgene ), the expression of the marker serving to indicate the presence of that transgene or its associated trait in an individual . this may be used as an indicator of successful transformation , or of the inheritance of the transgene in the progeny of a transformant ( i . e . in an insect line ). expression of the marker protein can be used to allow the assessment of the degree of inclusion of a transgene , or other effector , into the population . this has several advantages , including that , like any such marker , it identifies the presence of the transgene , so one can follow inheritance . the more tightly the marker is linked to the trait of interest e . g . a lethal system , the less likely it is that mutations occur which inactivate one but not the other . in practice , though , if the marker and transgene are on the same inserted dna segment , then this is extremely unlikely in any case . the polynucleotides making up the present expression system may be dna , rna or a mixture of both . the system may be inducible or it may be repressible . suitable examples exist in the art , including gal4 - uas or the tet - on or tet - off systems , described for instance in our ridl pct publication mentioned hereinabove . expression of the marker gene may therefore be induced or de - repressed , for instance by removal of tetracycline from the environment , such as the diet , of the insect . in a further aspect , a method of quality control is hereby provided , comprising expressing the present expression system in a target group of individuals and determining whether those individuals meet expected criteria such as size , number , developmental stage or localisation . for instance , if the marker gene is a reporter such as a fluorescent protein , then the individuals where expression from the system occurs will become visible under suitable wavelengths of light . this method may further comprise inducing or de - repressing expression of the present expression , for instance as mentioned above , so that the individuals where expression from the system has been induced or de - repressed will become visible under suitable wavelengths of light . although cytoplasmic actin promoters have been used in the past as transformation markers ( see above and below ), this is the first use of a muscle actin promoter being used for a similar purpose . furthermore , we have successfully utilised the same plasmid , # 4014 , fig2 , in two different tephritids ; the mediterranean fruit fly , c . capitata , and the olive fly , b . oleae , with similar transgenic insect phenotypes and levels of dsred2 expression . to our surprise , expression of the dsred2 fluorescent protein under the mexfly muscle actin promoter is so strong that transgenic individuals are easily recognizable by the naked eye at all larval , pupal and in some cases adult stages . moreover , despite the strong dsred2 expression , no adverse effects of the transgene have been encountered in any of the lines we have generated so far ; strains have been kept in our laboratories for more than 2 years . ox4676 was also used in both species . we have tested medfly adults containing either the ie1 - hr5 or polyubiquitin promoter sequences driving fluorescent proteins , in various simulated trapping conditions , under a number of different temperature and humidity environments , and found that although both markers express well in dead tissue for a week , the accuracy of identification between a transgenic and wild type insects drops considerably after longer periods on a trap . however , fluorescence was identified accurately in the same experiment , after a month in a trap , in medfly individuals carrying the mexfly - muscle actin promoter — dsred2 plasmid , # 4014 , fig2 . furthermore , in the latter case , medflies did not require removal from the trap , which is usually required , as commonly used yellow sticky traps exhibit quite a strong colouring under various excitation filters . owing to the conserved nature of the actin genes , similar , or the same , muscle actin promoters may be used other insects , for example lepidoptera , coleopterans and mosquito species , with minimal genetic manipulation . we have also isolated a muscle actin promoter from bombyx mori , and have prepared a gene expression system comprising the same . this gene expression system was tested in pink bollworm ( example 5 ). the results were comparable to medfly individuals transformed with # 4014 construct . moreover , we have also isolated a muscle actin promoter from aedes aegypti based on homology to published genome sequence . particularly preferred is a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly . a muscle actin promoter from other species may also be used and , in some embodiments , the origin of the promoter will tally with the species in which it is expressed ( i . e . the species to be transformed ). in other embodiments , a muscle actin promoter from one species may be used for expression of the fluorescent protein in another species . that other species may be in the same class ( i . e . another insect ), but more preferably in the same order or family , and most preferably in the same genus as the organism to be transformed . as such , a muscle actin promoter may be isolated from any insect , preferably from the order diptera or the order lepidoptera . more preferably , the muscle actin promoter may be from a culicidae and , most preferably , from a mosquito , preferably from the genera stegomyia , aedes , anopheles or culex . particularly preferred are stegomyia aegyptae , also known as aedes aegypti , stegomyia albopicta ( also known as aedes albopictus ), anopheles stephensi , anopheles albimanus and anopheles gambiae . it is also preferable that the muscle actin promoter is from a tephritid , preferably from medfly ( ceratitis capitata ), preferably from mexfly ( anastrepha ludens ), preferably from oriental fruit fly ( bactrocera dorsalis ), olive fruit fly ( bactrocera oleae ), melon fly ( bactrocera cucurbitae ), natal fruit fly ( ceratitis rosa ), cherry fruit fly ( rhagoletis cerasi ), queensland fruit fly ( bactrocera tyroni ), peach fruit fly ( bactrocera zonata ) caribbean fruit fly ( anastrepha suspensa ) or west indian fruit fly ( anastrepha oblique ). medfly ( ceratitis capitata ), mexfly ( anastrepha ludens ), or olive fruit fly ( bactrocera oleae ) are particularly preferred . within lepidoptera , moths are particularly preferred , including codling moth ( cydia pomonella ), and the silk worm ( bombyx mori ), the pink bollworm ( pectinophora gossypiella ), the diamondback moth ( plutella xylostella ), the gypsy moth ( lymantria dispar ), the navel orange worm ( amyelois transitella ), the peach twig borer ( anarsia lineatella ) and the rice stem borer ( tryporyza incertulas ), also the noctuid moths , especially heliothinae . the muscle actin promoter is also preferably from drosphila melanogaster . most preferably , it is that provided in seq id nos : 5 or 6 and most preferably seq id no : 1 or variants of any thereof . gene expression systems of the present invention , comprising a muscle actin promoter from the any of the above organisms , may be expressed in a range of insects . it is preferred that the gene expression system is capable of expression in dipterans and most preferably tephritids , medfly ( ceratitis capitata ), mexfly ( anastrepha ludens ), preferably oriental fruit fly ( bactrocera dorsalis ), olive fruit fly ( bactrocera oleae ), melon fly ( bactrocera cucurbitae ), natal fruit fly ( ceratitis rosa ), cherry fruit fly ( rhagoletis cerasi ), queensland fruit fly ( bactrocera tyroni ), peach fruit fly ( bactrocera zonata ) caribbean fruit fly ( anastrepha suspensa ) or west indian fruit fly ( anastrepha obliqua ). where a list of options is provided herein , such as the list of insect types in which it is preferred that the systems of the present invention can be expressed , it will be appreciated that the present invention contemplates each member of that list individually , as well as being part of that group . it is also particularly preferred that the gene expression system is capable of expression in a mosquito , preferably from the genera stegomyia , aedes , anopheles or culex . particularly preferred are stegomyia aegyptae , also known as aedes aegypti , stegomyia albopicta ( also known as aedes albopictus ), anopheles stephensi , anopheles albimanus and anopheles gambiae . expression in lepidoptera is also preferred , especially moths , including codling moth ( cydia pomonella ), and the silk worm ( bombyx mori ), the pink bollworm ( pectinophora gossypiella ), the diamondback moth ( plutella xylostella ), the gypsy moth ( lymantria dispar ), the navel orange worm ( amyelois transitella ), the peach twig borer ( anarsia lineatella ) and the rice stem borer ( tryporyza incertulas ), also the noctuid moths , especially heliothinae . in a further aspect , expression may be in a beneficial insect . a beneficial insect , in the context of the present invention , is , for example , a pollinator and / or is a predator or parasitoid , parasite suitable for controlling agricultural or horticultural pests . especially preferred are bees , including honeybees , bumblebees and other pollinators . some bees are pollinators , whilst others are predators or act as parasites . thus , predatory or parasitic bees and wasps may also be preferred beneficials . another preferred group of beneficials are ladybugs . the preferred beneficial can also be selected from the group consisting of : minute pirate bugs ; big eyed bugs ; assassin bugs ; damsel bugs ; mealybug destroyer ; soldier beetle ; green lacewing ; predatory mites , e . g . amblyseius swirskii , phytoseulius persimilis , typhlodromus pyri and amblyseius fallacis , syrphid fly ; tachinid fly ; ichneumon wasps , e . g . aphidius spp ., aphelinid wasps , e . g . encarsia spp . and trichogramma wasps . it is particularly preferred that the gene expression system comprises a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly , and can be expressed in any of the above insects . however , it will also be appreciated that any combination of the various promoters discussed herein can be used in any of the insects discussed herein . one particularly preferred example is the use of a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly in tephritids , especially fruit flies . another particularly preferred example is the use of a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly in mosquitoes , especially aedes , including aedes aegypti . another particularly preferred example is the use of a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly in moths , especially bombyx mori or plutella xylostella . another particularly preferred example is the use of a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly in medfly ( ceratitis capitata ). another particularly preferred example is the use of a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly in olive fly ( bactrocera oleae ). another particularly preferred example is the use of a muscle actin promoter from bombyx mori or aedes aegypti , and most preferably from mexfly in pink bollworm . the insect in which the present system is expressed may be referred to as a transformant , or may be considered to be the progeny thereof , or as a host . the insect may be a pest , for instance a disease vector , for instance a mosquito species that may carry the malarial parasite or act as a vector for dengue fever , or an agricultural pest , for instance moths or flies , especially fruit flies . the system described herein is suitable for expression in an insect , but it will be understood that this may also be preferably referred to as “ capable of expression in ” or “ adapted to or having the ability to express in an insect ”. it is preferable that insects are transformed with the gene expression system by injection into embryos . plasmids , or other suitable vectors , may be used as vehicles for the expression systems of the present invention , and may be introduced into the population by any suitable transformation means known in the art . a promoter is typically understood to be a region of dna sufficient to initiate transcription of a gene , and should preferably include features such as promoter elements such as transcription factor and rna polymerase binding sites . it may comprise further regulatory elements such as enhancers , and extended regions of dna for 5 ′ and 3 ′ utrs which enhance expression of the gene . the skilled person would be able to assess which features are necessary for improved expression of the gene . a particularly preferred 3 ′ utr is the 3 ′ utr of the muscle actin gene from anastrepha ludens . when a muscle actin promoter is referred to as being “ isolated from ”, “ taken from ” or simply “ from ” a particular species , it will be understood that it does not necessarily mean that the particular dna is derived from an organism belonging to that species . instead , it will be understood that it means that the dna sequence is largely , or completely , identical to that found in the genome of the wildtype of that species . the extraction or isolation may have occurred in the past and may have been used in the transformation of the host insect &# 39 ; s ancestors . the gene expression system comprises a muscle actin promoter “ operably linked ” to a marker gene . in this context , it will be understood that this means that the promoter is suitable or capable of driving transcription of the marker gene . it is generally preferred that they are situated substantially adjacent each other and not separated by more than 3 or 4 kbp . by “ gene ” herein is meant principally a sequence encoding an effector . in this case , the effector is largely a protein so as to function as a marker , as required , generally so as to express a detectable marker . preferably , the marker is a protein , which confers a detectable change in phenotype of the insect . a marker gene is a gene used to determine if a nucleic acid sequence has successfully transformed an organism such that it is being expressed . the nucleic acid sequence may , for example , encode a functional protein which causes sterility in an insect . the marker gene may be considered as a screening marker as opposed to a selectable marker , which may protect the organism from a selective agent that would normally kill it or prevent its growth , such antibiotic . a marker for screening will cause the cells containing the marker gene to appear different to those that , for instance , have not been transformed or which do not contain the transgene . a preferred example of a screening marker is a fluorescent protein , such as gfp . as such , it is preferable that the marker gene of the expression system described herein encodes a fluorescent protein . it is particularly preferred that the fluorescent protein is a green fluorescent protein ( gfp ), zsgreen , turbogfp , yellow fluorescent protein ( yfp ), mcitrine , dsred , dsred2 , mcherry , amcyan , cypet and other members of the gfp - like protein super - family . these proteins are typically derived from marine invertebrates , e . g . anemones , and when expressed emit fluorescence after exposure to bright light of a specific excitation wavelength . fluorescent proteins are widely used for transgenic research purposes . 3 in the red spectrum any of the following are preferred : dsred - express , tdtomato , dsred monomer , asred2 , mstrawberry , mcherry , mraspberry , e2 - crimson , or mplum . in the green spectrum , either of the following is preferred : egfp , acgfp1 . the marker gene may encode other proteins manifesting a phenotype which varies depending on the level of expression . this has the advantage of allowing qualitative or quantitative analysis for screening . where reference to a particular nucleotide sequence is made , it will be understood that this includes reference to any mutant or variant thereof , having substantially equivalent biological activity thereto . preferably , the mutant or variant has at least 85 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 %, preferably at least 99 . 9 %, and most preferably at least 99 . 99 % sequence identity with the reference sequences . we therefore report a unique marker system and methods for simple and reliable monitoring of released insects in the field that confers significant cost benefits to a sit control pest programme and requires minimal training of personnel . released males may also be identified by farmers and workers in the field with the use of a portable lamp with the appropriate excitation wavelengths , eliminating the need for the traps to be transferred to a laboratory facility for careful examination and identification of the trapped insects . also , the use of such a marker allows for the development of a highly accurate , automated detection and monitoring system based on computer software , which is able to detect the phenotype conferred by the marker gene of the expression system . mounier et al ( 1992 ) compared the sequences of insect muscle and cytoplasmic actins and identified bm a1 ( from bombyx mori ), dm act57a and dm act87e ( both from drosophila melanogaster ) as muscle actins . the cdna sequences of these 3 genes were aligned using clustalw , and the following degenerate primers designed : which amplify a 718 bp product . these primers were used for pcr on mexfly adult female cdna using phusion polymerase . the resulting pcr product was cloned and sequenced . the sequence ( confirmed by blast comparison to genbank sequences ) was extended by 5 ′ and 3 ′ race in order to obtain the complete mrna sequence . the promoter sequences was isolated from the coding sequence of the mexfly muscle actin gene using mexfly genomic dna was digested with clai , bamhi and sphi restriction enzymes . it was ligated to adaptors consisting of an appropriate sticky end for ligation to the digested dna and primer annealing sites for primers : pcr was carried out on these templates using 222 ) primer and 453 ) mexmufir . the resulting pcr product was diluted 1 in 100 then used as a template for nested pcr using primers 223 ) mid and 454 ) mexmufirn . these pcr products were cloned using the topo pcr cloning kit ( invitrogen ) and sequenced . were designed from this sequence to use on the same ligated dna templates using primer and mid primers as before . this extended the promoter sequence further . a 2 . 2 kb sequence incorporating the promoter and 5 ′ utr ( untranslated region ) ( fig1 ) were cloned in front of the start atg codon of dsred2 fluorescent protein ( fig1 ), in a plasmid (# 4104 , fig2 ) also containing piggy bac transposon ends and the transformation marker , hr5ie1 - zsgreen . # 4104 contains mexmuac promoter driving dsred2 fluorescent protein ; known transformation marker , hr5ie1 - zsgreen ; piggybac ends for transformation into insect cells ; sv40 3 ′ utr sequence ( fig1 ), bacterial origin of replication ( puc ori ) and ampicillin resistance gene ( bla ( amp [ r ])) for growth and selection in escherichia coli . regarding fig2 , ox4676 was used in both species . others were used for both olive fly and medfly with the single difference that we used 4 - ended piggybac constructs for medfly and single ended piggybac constructs for olive fly . despite the different numbers , all constructs utilised the exact same sequences relating to the transformation marker . this is the first demonstration of dsred2 expression under the mexfly muscle actin promoter . the plasmid utilises a green fluorescent protein ( zsgreen ; clontech ) under a generic promoter - enhancer sequence ; hr5 - ie1 as a transformation marker . as we did not know at that stage whether the isolated dna sequence upstream of the mexfly muscle actin gene served as an adequate promoter sequence to drive dsred2 expression , even more so in another tephritid ; ceratitis capitata , we thought it best to utilise a promoter sequence known to function well previously to drive the expression of a different fluorescent protein ; zsgreen in order to identify transformed individuals . both markers utilised the bi - directional sv40 3 ′ utr previously functional in a number of different insect species . 530 pre - blastoderm medfly embryos were injected with a piggybac transposon - transposase mix ( 600 : 300 ng / μl , respectively ). 260 g0 adults were back - crossed to wild type of the opposite sex , in pools of 10 . progeny was collected and screened for the presence of both fluorescent proteins . 8 independent insertion events were generated . all lines exhibited green fluorescence under the appropriate excitation wavelength as expected . furthermore , all lines displayed a strong red expression in all muscle tissue under the appropriate wavelength . fluorescence was clearly detectable in all developmental stages . no recordable differences were observed among siblings of the same strain , indicating a uniform and ubiquitous expression . red expression was so strong in most of the strains analysed that a pink hue was clearly visible at mid - late larval and early - mid pupal stages with naked eye ; that is transformed individuals were evident with 100 % accuracy without the need of a standard microscope equipped with the appropriate excitation filters for the fluorescent protein used ( dsred2 in this particular case ), a standard procedure for fluorescent screening . adult colouration was not apparent in any of these strains . nevertheless , all adults — at all ages — displayed a strong red expression when examined under a suitable microscope . two of these strains were tested in parallel with other medfly strains utilising hr5 - ie1 and polyubiquitin promoter sequences driving expression of dsred2 , for dead tissue fluorescence sustainability under various trapping methods and simulated environmental conditions and clearly outperformed existing markers . # 4014 medfly strains have been continuously reared in our premises for over 40 generations without an observable fitness penalty in adult longevity , female egg fecundity , embryo hatching or pupal recovery . subsequent sperm - lethal constructs (# 4676 , # 4705 # 4751 and # 4718 ) ( gb2500113 and wo2013 / 131920 ) used the mexfly muscle actin 3 ′ utr ( fig1 ), which resulted in stronger expression . therefore , one way of making the bombyx muscle actin promoter stronger , may be to incorporate its 3 ′ utr into the construct . given the desirable properties of the mexfly muscle actin promoter as a transformation marker and monitoring tool , we decided to further utilise this promoter in conjunction with our newly developed sperm lethal technology ( gb2500113 ) in this instance and in all examples following , we have utilised the 3 ′ utr of the muscle actin gene from anastrepha ludens . plasmid # 4751 was injected in preblastoderm medfly embryos as described above . two different transformation events were attained . both strains displayed robust red fluorescence under a suitable microscope and a “ light red ” phenotype clearly visible with naked eye at larval and pupal stages . in one of the two strains , visible colouration was apparent even at the adult stage , irrespective of the dark exoskeleton processed by this species . this may indicate an improved expression of the fluorescent protein possibly through utilisation of its own utr sequence . as before , no detrimental effects were observed in either strain for any rearing parameter . in most cases , an insect male has the capacity of mating more than one female whilst females of most species usually mate only once in their lifetime . this means that one could exploit double , triple or even multiple the amount of females , compared to the amount of males , in mass - rearing cages to increase egg yield and therefore production of males for release in a cost - effective manner . this is in operation in medfly mass - rearing factories today , utilising the white pupae mutation associated with the tsl ( temperature sensitive lethal ) sexing strains ; females are white as pupae compared to males that display the “ normal ” brown pupal phenotype . nevertheless , the white pupae mutation or any other phenotypic mutation that can be used in a similar way has to be isolated , characterised and cloned separately for all insect pests that sit can be applied to , a laborious and costly procedure . development of differential fluorescent expression in males and females is a far less strenuous , affordable and thus widely applicable way of achieving similar outcomes in a number of different insect species of interest , with none or minimal manipulation among species . to investigate this possibility , we have developed construct # 4676 where the mexfly muscle actin promoter drives expression of dsred2 in males and females but also drives expression of zsgreen in females only . for female specificity , we have utilised the same sex specific alternative splicing described and patented prior to this experiment ( pct / gb2007 / 000488 ). insertion of a cassette exon from the c . capitata transformer gene between the dsred2 and zsgreen dna sequences means that the zsgreen transcript is disrupted in male splice variants but not in the female - specific ones . this construct has been injected in medfly and olive fly pre - blastoderm embryos . 4 independent insertion events were generated in medfly and 7 in olive fly . males and females of all strains — both species — exhibited the red fluorescent phenotype , while females only of all strains displayed the green fluorescent phenotype under the appropriate excitation filters . in other words , transposition of this construct to two different tephritids resulted in the expected phenotype . nevertheless , none of the strains displayed the strong fluorescent expression seen previously with this promoter . this could be the result of a very long and thus unstable transcript being produced which may comprise expression levels . furthermore , green fluorescence was sometimes hard to detect in females of some strains — especially in medfly , possibly due to positional effects . this is a single piggybac construct utilising our novel “ sperm lethal ” technology discussed in example 2 in combination with our unique transformation marker promoter from a . ludens . the transposon was co - injected with piggybac transposase mrna ( 600 : 500 ng / μl ) into preblastoderm olive fly embryos . ten independent transgenic strains were generated all displaying a very strong and ubiquitous fluorescent phenotype under a suitable microscope and different hues of red colouring in larvae and pupae . bma1 promoter was identified by blast of the published coding sequence against the published bombyx mori genomic sequence ( whole genome sequencing project : http :// silkbase . ab . a . u - tokyo . ac . jp / cgi - bin / index . cgi ). a 2 . 58 kb fragment 5 ′ of the gene was amplified as the promoter fragment , from bombyx mori genomic dna ( fig3 ). using plasmid dna amplified and purified by midiprep or maxiprep , dna mixes were prepared to a final concentration of 500 ng / μl and 300 ng / μl of construct and helper plasmid ( making piggybac transposase ), respectively , after final re - suspension in injection buffer . 1467 embryos were injected with this construct (# 4143 , fig4 ), resulting in 238 survivors and 1 line . the green fluorescence was observed in transgenic insects . construct # 4143 ( fig4 ) consists of bombyx mori bma1 muscle actin promoter driving zsgreen fluorescent protein ; known transformation marker baculovirus opie2 promoter driving dsred2 ; pb transposon ends for transformation into insects ; bacterial origin of replication ( puc ori ) and ampicillin resistance gene ( bla ampr ) for growth and selection in e . coli . construct # 4247 was also injected ( fig5 ). the only difference between this and # 4143 ( fig4 ) is that there are an extra 340 nucleotides at the beginning of the promoter ( 5 ′ end ) and it also includes 66 bp comprising the 5 ′ utr at the 3 ′ end of the promoter ( which should make the promoter much more efficient ). 1945 embryos were injected with this construct resulting in 588 survivors . similarly , the published genome sequence from aedes aegypti ( http :// aaegypti . vectorbase . org /) was searched using blast for homology to the mexfly muscle actin gene . this identified aedes actin1 as the muscle actin gene . this gene has a large intron in the 5 ′ utr ( fig8 ), which was truncated in the construct (# 4133 , fig7 ). the promoter ( fig6 ) was amplified in two fragments using the following primer pairs : seq id no : 1 — mexfly muscle actin promoter and 5 ′ utr region used in construct # 4014 seq id no : 17 — 5 ′ utr ( including truncated intron in lower case ) of # 4133 allen , m ., handler , a ., berkebile , d ., and skoda , s . 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