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TRANSGENIC ANIMALS

INTRODUCTION: The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome - the material responsible for inherited characteristics - in contrast to spontaneous mutation (FELASA September 199 ! re"ised Februar# 199$%& Foreign '(A is introduced into the animal! using recombinant '(A technolog#! and then must be transmitted through the germ line so that e"er# cell! including germ cells! of the animal contain the same modified genetic material&

Historical background )rior to the de"elopment of molecular genetics! the onl# wa# of stud#ing the regulation and function of mammalian genes was through the obser"ation of inherited characteristics or spontaneous mutations& Long before *endel and an# molecular genetic +nowledge! selecti"e breeding was a common practice among farmers for the enhancement of chosen traits! e&g&! increased mil+ production& 'uring the 19,-s! the first chimeric mice were produced (.rinster! 19,/%& The cells of two different embr#os of different strains were combined together at an earl# stage of de"elopment (eight cells% to form a single embr#o that subse0uentl# de"eloped into a chimeric adult! e1hibiting characteristics of each strain& The mutual contributions of de"elopmental biolog# and genetic engineering permitted rapid de"elopment of the techni0ues for the creation of transgenic animals& '(A microin2ection! the first techni0ue to pro"e successful in mammals! was first applied to mice (3ordon and 4uddle! 1951% and then to "arious other species such as rats! rabbits! sheep! pigs! birds! and fish& Two other main techni0ues were then de"eloped: those of retro"irus-mediated transgenesis (6aenisch! 19,7% and embr#onic stem (ES% cell-mediated gene transfer (3ossler et al&! 1957%& Since 1951! when the term transgenic was first used b# 6&8& 3ordon and F&9& 4uddle (1951%! there has been rapid de"elopment in the use of geneticall# engineered animals as in"estigators ha"e found an increasing number of applications for the technolog#&

Met ods o! creation o! transgenic ani"als


For practical reasons! i&e&! their small si:e and low cost of housing in comparison to that for larger "ertebrates! their short generation time! and their fairl# well defined genetics! mice ha"e become the main species used in the field of transgenics& The three principal methods used for the creation of transgenic animals are '(A microin2ection! embr#onic stem cell-mediated gene transfer and retro"irus-mediated gene transfer& a# DNA "icroin$ection% This method in"ol"es the direct microin2ection of a chosen gene construct (a single gene or a combination of genes% from another member of the same species or from a different species! into the pronucleus of a fertili:ed o"um& ;t is one of the first methods that pro"ed to be effecti"e in mammals (3ordon and 4uddle! 1951%& The introduced '(A ma# lead to the o"er- or under-e1pression of certain genes or to the e1pression of genes entirel# new to the animal species& The insertion of '(A is! howe"er! a random process! and there is a high probabilit# that the introduced gene will not insert itself into a site on the host '(A that will permit its e1pression& The manipulated fertili:ed o"um is transferred into the o"iduct of a recipient female! or foster mother that has been induced to act as a recipient b# mating with a "asectomi:ed male& The first successful production of transgenic mice using pronuclear microin2ection was reported in 195- (3ordon et al&! 195-%& Although the recombinant "iral construct was pro"en to ha"e integrated into the mouse genome! it was rearranged and did not e1press& Subse0uent reports (.rinster et al&! 1951< =ostantini and Lac#! 1951% pro"ed that integrated transgenes were capable of functional e1pression following pronuclear microin2ection& The first "isible phenot#pic change in transgenic mice was described in 195 for animals e1pressing the rat growth hormone se0uence ()almiter et al&! 195 %& =urrentl#! se"eral hundred transgenic e1pression papers are published each #ear! the ma2orit# e1amining the effects of microin2ected "iral se0uences on mammalian growth and patholog#& The pronuclear microin2ection method of producing a transgenic animal results in the introduction of a purified double-stranded '(A se0uence into the chromosomes of the fertili:ed mammalian egg& ;f this transferred genetic material (i&e&! transgene% is integrated into one of the embr#onic chromosomes! the animal will be born with a cop# of this new information in e"er# cell& The foreign '(A must integrate into the host genome prior to the doubling of genetic material that precedes the first clea"age or a mosaic animal ma# be produced in which man# cells do not possess the new gene& For this reason! the transgene '(A is introduced into the :#gote at the earliest possible stage! i&e&! the pronuclear period immediatel# following fertili:ation& For se"eral hours following the entr# of the sperm into the ooc#te! the male and female pronuclei are microscopicall# "isible as indi"idual structures& The transgene ma# be microin2e cted into either of these pronuclei with e0ui"alent results& 9owe"er! >-chromosome or ?-chromosome integration e"ents do occur and ob"iousl# ma# be influenced b# the choice of pronucleus& @suall#! the male pronucleus ma# be distinguished because it is larger than the female nucleus and also because it is closer to the ooc#te surface&

The animal that de"elops after recei"ing the transgene '(A is referred to as the founder (Fo% of a new transgenic lineage& ;f the germ cells of the founder (mosaic or not% transmit the transgene stabl#! then all descendants of this animal are members of a uni0ue transgenic lineage& ;ntegration of foreign '(A into the embr#onic genome generall# is a random e"ent with respect to the chromosomal locus& Therefore the probabilit# of identical integration e"ents in two embr#os recei"ing the same transgene is o"erwhelmingl# unli+el#& ;n addition! it is impossible to regulate e1actl# how man# copies of the transgene will be introduced into the embr#o and how man# will 2oin together to integrate (usuall# at a single site% as a single linear arra# called a concatamer (.rinster et al&! 1951! 195$< .ishop and Smith! 1959%& *an# studies ha"e found dramatic differences in the e1pression of a specific transgene within indi"idual sibling embr#os simpl# due to different integration loci& The num ber of copies of the
transgene that ha"e 2oined the founderAs genome is referred to as the copy number, and rarel# appears to be correlated with the degree of transgene e1pression in the animal&

.ecause the locus of transgene integration is random! the transgene fre0uentl# inserts into functional genetic se0uences& ;nterruption of the normal e1pression of an endogenous gene ma# be inconse0uential or lethal& Alternati"el#! obser"able insertional mutagenesis might be apparent when the insertion interferes with the e1pression of an endogenous de"elopmentall# acti"e gene& These mutations are distinguished from the true transgenic phenot#pe because onl# a single lineage e1hibits the defect& The mutations can in"ol"e an# s#stem including the special senses! cardio"ascular! neurological and reproducti"e s#stems! and se"ere morphogenetic abnormalities ma# be obser"ed (8o#chi+ et al&! 195$%& The identification of the locus of transgene insertion is of great "alue because it maps the locus of an important endogenous gene& .ecause the new transgenic locus is present in onl# one member of a particular paired chromosome! the genot#pe of the founder is described as hemizygous for the transgene rather than hetero:#gous& A homo:#gous genot#pe! in which a pair of transgene alleles is present! ma# be produced b# the mating of a pair of hemi:#gous F1 siblings& Bb"iousl#! mating a pair of animals with identical transgenes but from different founder lineages cannot result in a true homo:#gote in which independe nt segregation of the loci is predictable& The success of the microin2ection techni0ue relies upon the careful collection of a relati"el# large group of accuratel# timed embr#os from a reproducti"el# s#nchroni:ed group of female embr#o donors& ;n addition! the techni0ues of microin2ection and embr#o transfer to a suitable recipient female must be mastered& Bf course! the combined success of all of these manipulati"e s+ills ultimatel# depends upon the fastidious construction and preparation of the transgene '(A fragments to be in2ected&

A ma2or ad"antage of this method is its applicabilit# to a wide "ariet# of species&

b# E"br&onic ste" cell'"ediated gene trans!er% This method in"ol"es prior insertion of the desired '(A se0uence b# homologous recombination into an in "itro culture of embr#onic stem (ES% cells& Stem cells are undifferentiated cells that ha"e the potential to differentiate into an# t#pe of cell (somatic and germ cells% and therefore to gi"e rise to a complete organism& These cells are then incorporated into an embr#o at the blastoc#st stage of de"elopment& The result is a chimeric animal& ES cell-mediated gene transfer is the method of choice for gene inacti"ation! the socalled +noc+-out method& This techni0ue is of particular importance for the stud# of the genetic control of de"elopmental processes& This techni0ue wor+s particularl# well in mice& ;t has the ad"antage of allowing precise targeting of defined mutations in the gene "ia homologous recombination

METHOD(

)% Make &our DNA @sing recombinant '(A methods! build molecules of '(A containing

the structural gene #ou desire (e&g&! the insulin gene% *ector '(A to enable the molecules to be inserted into host '(A molecules +ro"oter and en ancer se,uences to enable the gene to be e1pressed b# host cells

-% Trans!or" ES cells in culture E1pose the cultured cells to the '(A so that some will incorporate it& .% Select !or success!ull& trans!or"ed cells% /% In$ect t ese cells into t e inner cell "ass 0ICM# o! "ouse blastoc&sts% 1% E"br&o trans!er

)repare a +seudo+regnant mouse (b# mating a female mouse with a "asectomi:ed male%& The stimulus of mating elicits the hormonal changes needed to ma+e her uterus recepti"e& Transfer the embr#os into her uterus& 9ope that the# i"+lant successfull# and de"elop into health# pups (no more than one-third will%&

2% Test er o!!s+ring

4emo"e a small piece of tissue from the tail and e1amine its '(A for the desired gene& (o more than 1-C -D will ha"e it! and the# will be hetero:#gous for the gene&

3% Establis a transgenic strain


*ate two hetero:#gous mice and screen their offspring for the 1:/ that will be o"o4&gous for the transgene& *ating these will found the transgenic strain&

c# Retro*irus'"ediated gene trans!er% A retro"irus is a "irus that carries its genetic material in the form of 4(A rather than '(A& This method in"ol"es: 7

retro"iruses used as "ectors to transfer genetic material into the host cell! resulting in a chimera! an organism consisting of tissues or parts of di"erse genetic constitution chimeras are inbred for as man# as - generations until homo:#gous (carr#ing the desired transgene in e"er# cell% transgenic offspring are born

The method was successfull# used in 19,/ when a simian "irus was inserted into mice embr#os! resulting in mice carr#ing this '(A& To increase the probabilit# of e1pression! gene transfer is mediated b# means of a carrier or "ector! generall# a "irus or a plasmid& 4etro"iruses are commonl# used as "ectors to transfer genetic material into the cell! ta+ing ad"antage of their abilit# to infect host cells in this wa#& Bffspring deri"ed from this method are chimeric! i&e&! not all cells carr# the retro"irus& Transmission of the transgene is possible onl# if the retro"irus integrates into some of the germ cells& For an# of these techni0ues the success rate in terms of li"e birth of animals containing the transgene is e1tremel# low& )ro"iding that the genetic manipulation does not lead to abortion! the result is a first generation (F1% of animals that need to be tested for the e1pression of the transgene& 'epending on the techni0ue used! the F1 generation ma# result in chimeras& 8hen the transgene has integrated into the germ cells! the so-called germ line chimeras are then inbred for 1- to - generations until homo:#gous transgenic animals are obtained and the transgene is present in e"er# cell& At this stage embr#os carr#ing the transgene can be fro:en and stored for subse0uent implantation& Transgenic Ani"als as 5iotec nolog& Transgenic animals are 2ust one in a series of de"elopments in the area of biotechnolog#& .iotechnolog# has transformed the wa# in which we understand processes such as engineering and manufacturing& These terms now include the use of li"ing organisms or their parts to ma+e or modif# products! to change the characteristics of plants or animals! or to de"elop micro-organisms for specific uses& The no"el uses of biological techni0ues such as recombinant '(A techni0ues! cell fusion techni0ues! mono and pol#clonal antibod# technolog# and biological processes for commercial production ha"e altered traditional distinctions and methods (@S =ongress! Bffice of Technolog# Assessment! 1959%& 3enetic manipulations at the le"el of '(A ha"e also changed long held "iews as to what is considered to be animal! plant and human& ;n turn! these changes ha"e made it more difficult to e"aluate the wa#s in which animals are used and ha"e obscured distinctions between pure and applied research& =onsideration of the acceptabilit# of creating specific transgenic animal strains or genetic manipulation in"ol"ing interchanging '(A between species and +ingdoms could be a simple animal care issue or a societal decision& The following is an attempt to show what the abilit# to create transgenic animals or engage in other forms of '(A manipulation means in terms of traditional A== functions! not forgetting that this impacts on wider considerations of human responsibilit# for the welfare of other life forms&

The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species! and is also affecting the numbers of animals used& This shift in the patterns of animal use is being monitored b# the ==A= through the use of the Animal @se 'ata Form& An e1ample of the replacement of higher species b# lower species is the possibilit# to de"elop disease models in mice rather than using dogs or non-human primates& ;n the long term! a reduction in the number of animals used! for e1ample to stud# human diseases! is possible due to a greater specificit# of the transgenic models de"eloped& Bn the other hand! the success of the method has led to using its potential for in"estigating a wider range of diseases and conditions& The actual use of some species ma# be increased! in addition to the numbers of animals which are sacrificed as donors during the creation process& The potential of the technolog# has also made it possible to consider emplo#ing cattle! swine! sheep and goats as processing units to manufacture proteins or as organ donors& The comple1 interacti"e processes of li"ing mammals are not reproducible in "itro& 9owe"er! transgenic animals pro"ide a means of e"aluating genetic modifications in terms of anatomical and ph#siological changes in a comple1 s#stem& Transgenic models are more precise in comparison to traditional animal models! for e1ample the oncomouse with its increased susceptibilit# to tumor de"elopment enables results for carcinogenicit# studies to be obtained within a shorter time-frame! thus reducing the course of tumor de"elopment in e1perimentall# affected animals& 9owe"er! models are not strict e0ui"alents! so as with an# other s#stem care must be ta+en in drawing conclusions from the data& A representati"e! but non-inclusi"e! list of purposes for which transgenic animals ha"e been used indicates the wide ranging application of this biotechnolog#:

in medical research! transgenic animals are used to identif# the functions of specific factors in comple1 homeostatic s#stems through o"er- or under-e1pression of a modified gene (the inserted transgene%< in to1icolog#: as responsi"e test animals (detection of to1icants%< in mammalian de"elopmental genetics< in molecular biolog#! the anal#sis of the regulation of gene e1pression ma+es use of the e"aluation of a specific genetic change at the le"el of the whole animal< in the pharmaceutical industr#! targeted production of pharmaceutical proteins! drug production and product efficac# testing< in biotechnolog#: as producers of specific proteins< geneticall# engineered hormones to increase mil+ #ield! meat production< genetic engineering of li"estoc+ and in a0uaculture affecting modification of animal ph#siolog# andEor anatom#< cloning procedures to reproduce specific blood lines< and de"eloping animals speciall# created for use in 1enografting&

;mportant general considerations include the e1tent to which e1perience ac0uired in the laborator# with regard to husbandr# should influence industr# standards for +eeping animals created specificall# as li"ing machines for the production of proteins! antibodies! etc& The successful cloning of 'oll# underlines the fact that inno"ati"e de"elopments in animal science are part of the mainstream of biotechnolog#& ;n addition! the use of 1enografts! at least at the public health le"el ma+es animal and human welfare inseparable&

A))L;=AT;B(S:
.reeding Farmers ha"e alwa#s used selecti"e breeding to produce animals that e1hibit desired traits (e&g&! increased mil+ production! high growth rate%&11!1$!1, Traditional breeding is a time-consuming! difficult tas+& 8hen technolog# using molecular biolog# was de"eloped! it became possible to de"elop traits in animals in a shorter time and with more precision& ;n addition! it offers the farmer an eas# wa# to increase #ields& Fualit# Transgenic cows e1ist that produce more mil+ or mil+ with less lactose or cholesterol1 ! pigs and cattle that ha"e more meat on them5!1,! and sheep that grow more wool15& ;n the past! farmers used growth hormones to spur the de"elopment of animals but this techni0ue was problematic! especiall# since residue of the hormones remained in the animal product 'isease resistance Scientists are attempting to produce disease-resistant animals! such as influen:a-resistant pigs! but a "er# limited number of genes are currentl# +nown to be responsible for resistance to diseases in farm animals& >enotransplantation )atients die e"er# #ear for lac+ of a replacement heart! li"er! or +idne#& For e1ample! about $!--organs are needed each #ear in the @nited Gingdom alone& $ Transgenic pigs ma# pro"ide the transplant organs needed to alle"iate the shortfall&9 =urrentl#! 1enotransplantation is hampered b# a pig protein that can cause donor re2ection but research is underwa# to remo"e the pig protein and replace it with a human protein&

(utritional supplements and pharmaceuticals )roducts such as insulin! growth hormone! and blood anti-clotting factors ma# soon be or ha"e alread# been obtained from the mil+ of transgenic cows! sheep! or goats& 4esearch is also underwa# to manufacture mil+ through transgenesis for treatment of debilitating diseases such as phen#l+etonuria ()G@%! hereditar# emph#sema! and c#stic fibrosis& ;n 199,! the first transgenic cow! 4osie! produced human protein-enriched mil+ at &/ grams per litre& This transgenic mil+ is a more nutritionall# balanced product than natural bo"ine mil+ and could be gi"en to babies or the elderl# with special nutritional or digesti"e needs& 4osieHs mil+ contains the human gene alpha-lactalbumin&

9uman gene therap# 9uman gene therap# in"ol"es adding a normal cop# of a gene (transgene% to the genome of a person carr#ing defecti"e copies of the gene& The potential for treatments for the $!--- named genetic diseases is huge and transgenic animals could pla# a role& For e1ample! the A& ;& Iirtanen ;nstitute in Finland produced a calf with a gene that ma+es the substance that promotes the growth of red cells in humans&

;n --1! two scientists at (e1ia .iotechnologies in =anada spliced spider genes into the cells of lactating goats& The goats began to manufacture sil+ along with their mil+ and secrete tin# sil+ strands from their bod# b# the buc+etful& .# e1tracting pol#mer strands from the mil+ and wea"ing them into thread! the scientists can create a light! tough! fle1ible material that could be used in such applications as militar# uniforms! medical microsutures! and tennis rac+et strings&1 To1icit#-sensiti"e transgenic animals ha"e been produced for chemical safet# testing& *icroorganisms ha"e been engineered to produce a wide "ariet# of proteins! which in turn can produce en:#mes that can speed up industrial chemical reactions

T e 6alue o! Transgenic Ani"als Transgenic animal s#stems combine the "irtues of cell culture and congenic breeding strategies while a"oiding the negati"e aspects of each s#stem& @sing transgenic techni0ues! a characteri:ed genetic se0uence ma# be e"aluated within the specific genomic bac+ground of the whole animal& Therefore! transgenic animals ma# be utili:ed to stud# the regulation of a specific genetic se0uence in a realistic fashion& *an# uses ha"e been de"eloped and man# more are forecast! particularl# in three areas: Models of human disease processes& 9undreds of transgenic rodent lines ha"e been produced b# introducing into the genome genetic se0uences such as "iral transacti"ating genes and acti"ated oncogenes implicated in specific pathologies& The phenot#pe and regulator# parameters of the gene then ma# be e"aluated in an animal model with a relati"el# short generation time& Also! normal rodent genetics and ph#siolog# are highl# characteri:ed& The predictabilit# of man# transgenic phenot#pes permits the i nno"ati"e testing of diagnostic and therapeutic agents while using a reduced population of e1perimental animals& The generation of no"el cell lines from transgenic organs also promises to reduce the number of research animals re0uired to e"aluate a therapeutic compound& ;n addition! transgenic genomes ma# be created in which more than one transgene ma# interact! or in which a transgene ma# interact with an endogenous normal or mutated gene& The use of transgenic disease models in biomedical research promises to accelerate dramaticall# the de"elopment of new human diagnostic and therapeutic treatments& Transgenic rodent models ha"e been characteri:ed for se"eral human diseases including cardio-"ascular disease (8alsh et al&! 199-%! cancer (Sinn et al&! 195,%! autoimmune disease (9ammer et al&! 199-%! A;'S (Iogel et al&! 1955%! sic+le cell anemia (4#an et al&! 199-% and neurological disease (Small et al&! 1957%& Targeted production of pharmaceutical proteins& Another use for transgenic animals in"ol"es the biological production of "aluable human protein en:#mes! hormones and growth factors&

These products ma# be recombinant or mutated! and collection of the functional protein from the animal emplo#s tissue-specific regulator# '(A se0uences! a strateg# described below& =urrent techni0ues in the biotechnolog# industr# use large-scale cell cultures to generate products in biological s#stems& Eu+ar#otic cell s or bacteria which ha"e ta+en up genetic e1pression se0uences (or constructs% are cultured in nutrient medium which is continuall# replaced and from which the bioengineered product is refined& This medium must be correctl# buffered and must be temperature-regulated and maintained pathogen-free& The use of transgenic animals! particularl# larger mammals! as bioreactors (Jpharmaceutical pharmingJ% is a cost-effecti"e alternati"e to cell culture methods& Animals automaticall# supplement thei r bodil# fluids with fresh nutrients! remo"e waste products! reliabl# regulate their internal temperature and p9 and resist pathogens& .# directing (or targeting% the e1pression of the transgene product so that it is produced b# the secretor# cells of the li"er! lactating mammar# gland or +idne#! JpharmersJ ma# collect and process bodil# fluids with minimal effort& The mammar# gland probabl# is the most promising target tissue because it produces large amounts of protein in a temperature-r egulated fluid that ma# be collected dail# in a non-in"asi"e fashion& Transgenic animals are not onl# cost-effecti"e bioreactors but! with the comple1 secretor# cell t#pes and organs of the mammalian organism! can perform much more complicated protein modifications than simpl# cultured cells Modification of animal anatomy and physiology& The most contro"ersial aspect of transgenic animal usage in"ol"es the Jselecti"e impro"ementJ of species b# the modification of the genome& *ost often! foreign genes are added to the host genome! but selecti"e deletion of specific genes or regions has been attempted& ;t has become apparent that merel# adding genes for growth factors or hormones to the genome is a simplistic approach to altering the comple1 multigenic ph#siolog# of the mamma l& The goals of this t#pe of e1periment ma# include decreased bod# fat! increased speed! no"el disease resistance or higher #ields of meat or mil+& At present! these t#pes of phenot#pic alterations are more realisticall# achie"ed in plants and bacteria than in animals&

ETHICAL ISSUES( Et ical Issues Related to Transgenic Ani"als - The social opinion on transgenic animal research is di"ided almost in the middle& Bpinion sur"e#s in @SA! 6apan and (ew Kealand re"eal that onl# / ! $/ and $5D! respecti"el#! of the people participating in the sur"e# fa"our such research& The main reasons for opposition of people is as follows&

Should there be uni"ersal protocols for transgenesisL Should such protocols demand that onl# the most promising research be permittedL ;s human welfare the onl# considerationL 8hat about the welfare of other life formsL Should scientists focus on in vitro (cultured in a lab% transgenic methods rather than! or before! using li"e animals to alle"iate animal sufferingL 8ill transgenic animals radicall# change the direction of e"olution! which ma# result in drastic conse0uences for nature and humans ali+eL Should patents be allowed on transgenic animals! which ma# hamper the free e1change of scientific researchL

1& @se of animals in biotechnological research causes great suffering to the animals& .ut most people seem to accept some animal suffering to ser"e the basic interest and welfare of man+ind< this attitude has been termed as interest-sensiti"e speciesism& & ;t is felt that b# using animals for the production of pharmaceutical proteins we reduce them to mere factories& This seems not to recognise that animals also are li"ing beings which feel pleasure and pain 2ust as we do& M& Some people feel that animals should be regarded as e0ual to humans in that the# ha"e the same basic rights as human beings& 9owe"er! in most societies animals are relegated to a position se"eral steps below that of man& /& An argument attempts to focus on integrit# of species in that each biological species has a right to e1ist as a separate identifiable entit#& .ut biologists do not regard a species as a fi1ed! water-tight entit#< rather the# are regarded as d#namic! constantl# e"ol"ing groups& $& Finall#! the introduction of human genes into animals! and "ice "ersa! ma# be seen b# man# as clouding the definition of JhumannessJ& .ut most of the +nown human genes are not uni0ue! and comparable genes do occur in animals& ;n addition! man# retro"iruses ha"e integrated into the human genome without an# recognisable de"aluation of our humanness&

Conclusion
;nterestingl#! the creation of transgenic animals has resulted in a shift in the use of laborator# animals N from the use of higher-order species such as dogs to lower-order species such as mice N and has decreased the number of animals used in such e1perimentation! 7 especiall# in the de"elopment of disease models& This is certainl# a good turn of e"ents since transgenic technolog# holds great potential in man# fields! including agriculture! medicine! and industr#&

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