ELSEVIER TUTORIAL Altering Cardiac Function via Transgenesis A Nuts and Bolts Approach Jeffrey Robbins Transgenesisprovides a means to modify the mammalian genome. By diyectingexpression of a engineered pr-oteinto the heart, one now is able to remodel effectively the cardiac protein profile and study the conse- quences of a single genetic manipulation at the moleculav biochemical, cytological, and physiologic levels. Often, a payticularpatho logy or even a global remodelingprocess such as hypertrophy is accompanied by the upregulation or downregulation of a gene or set(s) ofgenes. What is not known is whether these changes represent a beneficial compensato~ response or contribute to the continued degene~ation of normal heart function. The ability tope+orm genetic manipulations on cardiac gene expr-ession via transgenesis offers one a rapid and effective means of extending the correlations noted to the mechanistic level. Now, one can, in theory, ezp~ess a candidate protein at a particular developmental time and deter-mine the direct consequences of its appearance. Simi- larly, one can explore structure–function relationships, both between different forms of a protein family and in terms of active domains within a protein, by expressing a transgene that encodes a suitable mutation or ectopic protein isoform. This review explores the practical considerations of the transgenic approach in terms of what is impor- tant for a successful experiment from the necessary animal husbandry to designing constructs that will express at appropriate levels in the heart. (Trends Cardiovasc Med 1997; 7:185-191). 0 1997, Elsevier Science Inc. It is a basic tenet of modern biology that specific protein complements underliethe function of a cell or organ. Protein pro- duction is, in turn, ultimately controlled by cell-specific, defined transcriptional Jeffrey Robbinsis at theDivisionof Molecu- lar Cardiovascular Biology,Children’sHospi- tal Research Foundation, Cincinnati, OH 45229-3039, USA. patternsof the common gene set. Because cellular and, ultimately, organ function depend on the polypeptides that are present, it is not surprising that when function is altered, for example, during the development of disease, alterationsin the protein pools also take place. In the heart, numerous examples of protein changes correlated with functional alter- ations have been noted, both during nor- mal development and during the develop- ment of numerous pathologies [reviewed by Anversaet al. 1992, Figueredo and Ca- macho 1995,Francis et al. 1995, Payne et al. 1995,Blaufarb and Sonnenblick 1996). For example, one of the earliest correla- tions thatwas rigorously documented was the association of ~-myosin heavy chain (MyHC) gene (13-MYHC) expression in the ventricles of large animals, versus expres- sion of u-A4yHC in the ventriclesof smaller animals whose heart rates are faster (Barany 1967).Intuitively,this is a satisfy- ing observation, as the intrinsic ATPase activity of a-MyHC’s encoded protein is almost three times faster.Similarly,differ- ent congenital heart diseases are charac- terized by certain shifts in the motor pro- teins (Morano et al. 1996), and in heart failure, the upregulation or downregula- tion of different (hypothesized) effecters or modulators has been well documented in a number of models (Arai et al. 1993, Boluyt et al. 1994). What has been lacking for the major- ity of these observations is the extension of correlation to causative proof. To es- tablish cause, it is necessary to direct the heart t.o synthesize, in the absence of other pleiotropic changes, the candidate protein and subsequently to determine whether the protein’s presence causes the effects either directly or indirectly. By affecting the heart’s protein comple- ment in a defined manner, one has the means to establish both mechanism and the function of different proteins or pro- tein isoforrns. An additional level of res- olution can be applied to a particular protein. By judicious use of specific mu- tations in the sequences encoding sus- pected critical domains of a protein, par- tial or complete ablation of protein function can be achieved (a “dominant negative”). Alternatively,structure-func- tion relationships can be determined for the different domains within the whole organ and animal contexts. The scope of this review is not directed at the existing transgenic literature as it TCM vol.7, No. 6, 1997 @1997, Elsevier Science Inc., 1050-1738/97/$17.00 PII S105O-1738(97)OOO48-O 185
Transcript
ELSEVIER
TUTORIAL
Altering Cardiac Functionvia TransgenesisA Nuts and Bolts ApproachJeffrey Robbins
Transgenesisprovides a means to modify the mammalian genome. Bydiyectingexpression of a engineeredpr-oteinto the heart, one now is ableto remodel effectively the cardiac protein profile and study the conse-quences of a single genetic manipulation at the moleculav biochemical,cytological, andphysiologic levels. Often, apayticularpatho logy or evena global remodelingprocess such as hypertrophy is accompanied by theupregulation or downregulation of a gene or set(s) ofgenes. What is notknown is whether these changes represent a beneficial compensato~response or contribute to the continued degene~ation of normal heartfunction. The ability tope+orm genetic manipulations on cardiac geneexpr-ession via transgenesis offers one a rapid and effective means ofextending the correlations noted to the mechanistic level. Now, one can,in theory, ezp~ess a candidate protein at a particular developmentaltime and deter-mine the direct consequences of its appearance. Simi-larly, one can explore structure–function relationships, both betweendifferent forms of a protein family and in terms of active domainswithin a protein, by expressing a transgene that encodes a suitablemutation or ectopic protein isoform. This review explores the practicalconsiderations of the transgenic approach in terms of what is impor-tant for a successful experiment from the necessary animal husbandryto designing constructs that will express at appropriate levels in theheart. (Trends Cardiovasc Med 1997; 7:185-191). 0 1997, ElsevierScience Inc.
It is a basic tenet of modern biology thatspecific protein complements underliethefunction of a cell or organ. Protein pro-duction is, in turn, ultimately controlledby cell-specific, defined transcriptional
Jeffrey Robbinsis at the Divisionof Molecu-larCardiovascularBiology,Children’sHospi-tal Research Foundation, Cincinnati, OH45229-3039,USA.
patternsof the common gene set.Becausecellular and, ultimately, organ functiondepend on the polypeptides that arepresent, it is not surprising that whenfunction is altered, for example, duringthe development of disease, alterationsinthe protein pools also take place. In theheart, numerous examples of proteinchanges correlated with functional alter-ations have been noted, both during nor-
mal development and during the develop-ment of numerous pathologies [reviewedby Anversaet al. 1992,Figueredo and Ca-macho 1995,Francis et al. 1995,Payne etal. 1995,Blaufarb and Sonnenblick 1996).For example, one of the earliest correla-tions thatwas rigorously documented wasthe association of ~-myosin heavy chain(MyHC) gene (13-MYHC)expression in theventricles of large animals, versusexpres-sion of u-A4yHCin theventriclesof smalleranimals whose heart rates are faster(Barany 1967).Intuitively,this is a satisfy-ing observation, as the intrinsic ATPaseactivity of a-MyHC’s encoded protein isalmost three times faster.Similarly,differ-ent congenital heart diseases are charac-terized by certain shifts in the motor pro-teins (Morano et al. 1996), and in heartfailure, the upregulation or downregula-tion of different (hypothesized) effectersor modulators has been well documentedin a number of models (Arai et al. 1993,Boluyt et al. 1994).
What has been lacking for the major-ity of these observations is the extensionof correlation to causative proof. To es-tablish cause, it is necessary to direct theheart t.o synthesize, in the absence ofother pleiotropic changes, the candidateprotein and subsequently to determinewhether the protein’s presence causesthe effects either directly or indirectly.By affecting the heart’s protein comple-ment in a defined manner, one has themeans to establish both mechanism andthe function of different proteins or pro-tein isoforrns. An additional level of res-olution can be applied to a particularprotein. By judicious use of specific mu-tations in the sequences encoding sus-pected critical domains of a protein, par-tial or complete ablation of proteinfunction can be achieved (a “dominantnegative”). Alternatively,structure-func-tion relationships can be determined forthe different domains within the wholeorgan and animal contexts.
The scope of this review is not directedat the existing transgenic literature as it
applies to alteration of cardiac function;a number of recent reviews (Field 1993,Rubin and Smith 1994, Liu et al. 1995,Chien 1996) adequately outline the con-tributions these approaches have madein elucidating facets of normal and ab-normal heart development and function,and the field continues to develop rap-idly. Although other transgenic animalsexist, this review deals solely with themouse as a model system. The mouseremains the most practical model fortransgenic modulation of the genornebecause of its size (-23–30 g), gestationlength (21 days), time to sexual maturity(7 weeks), and the relative ease and costof the animal husbandry involved. Thetutorial will focus on those componentsnecessary for the effective remodeling ofthe mammalian heart using transgenesisand experimental considerations thatmay increase the chances of success.
. Feasibility
Assuming that technical considerationsare not rate limiting, the feasibility ofcarrying out a transgenic approach tothe chosen problem must be consideredcarefully. The animal husbandry, whilestraightforward, is often more costly andpersonnel intensive than first antici-pated. If the institution offers the ser-vices of a transgenic facility, this obvi-ously increases the feasibility of thegeneral approach for the individual in-vestigator. Usually, these facilities em-ploy the necessary rigor for handling ofthe animals in a barrier facility. The useof “clean animals,” that is, virus andpathogen free, is emphatically recom-mended. If the institution does not offerthe service, often productive collabora-tions can be formed with committed in-vestigators at other locations who haveaccess to the technology. A commercialconcern, DNX Transgenics (http:llww-w.dnxtrans.com/mouse. htm), also offersthe service, although it is relativelycostly and only a limited number offounders (three) are guaranteed.
The cost of the experiment will obvi-ously vary depending on the investiga-tor’s location but will be a function ofthe injection costs, institutional costs foranimal husbandry, the number of ani-mals needed, and the length of time thecolony will be kept at a high census forthe necessa~ analyses. We have found agood rule of thumb is that a single trans-
genic construct, from start to finish, willcost between $10,000 and $20,000 if acomplete set of molecular and physio-logic analyses over the course of a yearare carried out.
● Transcription:Choosinga Promoter
A critical consideration in building atransgene that will be expressed at highcardiac-specific levels is the choice of asuitable promoter. Early transgenic ex-periments used sequences such as thecytoplasmic actin or cytomegaloviruspromoters that drove expression in anumber of tissue types, including theheart. For cardiac-specific remodeling,however, it is advantageous to use amore specific promoter in order to avoidthe confounding effects of transgene ex-pression in other organs or tissues. Pref-erably, the chosen transcriptional appa-ratus should drive high levels of cardiac-specific manner at appropriate timesduring development and display a mini-mum of position-dependent effects; thatis, even though the transgenic constructinserts randomly into the genome andthus finds itself in different chromo-somal domains, expression levels arepartially or completely “insulated” fromany context effects.
The identification of cardiac-specifictranscriptional regulatory elements wasinitiatedwith the use of standard in vitroapproaches that centered around thetransection of various muscle cell lines.These experiments were only partiallysuccessful in identifyingthe required ele-ments because a true cardiogenic cell linedoes not existand because of the inherentlimitations of an in vitro environment, inwhich the transected regulatoryelementsare not integrated into the chromosome.Thus, more often than not, promotersclassifiedas “active”on thebasisof in vitroexperiments led to little or no detectableexpression in the animals’ hearts, whenused in transgenicanimals (Buttricket al.1993).In the last5 years,however,a num-ber of cardiac-specific promoters havebeen well characterized, not only in vitrobut also in vivo, eitherby injection directlyinto the heart (Molkentin and Markham1994)or by testingthe sequencesin trans-genic mice (Milano et al. 1994a): Thus,sequences derived from the cardiac regu-latory myosin light chain 2ven,nC1.(MLC2V) (Hunter et al. 1995), cardiac
a-actin (Biben et al. 1996), atrial natri-uretic factor (Field 1988) and a- and~-MyHC promoters (Rindt et al. 1995,Knotts et al. 1996) have all been used todrive gene expression in transgenic mice.
Many of these initial transgenic exper-iments used a “reporter gene” such as13-galactosidase or chloramphenicolacetyl transferase so that expressioncould be detected easily. This approachin the early stages of promoter identifi-cation is useful in that the onset andrelative degree of appropriate cardiacexpression, as well as ectopic expressionin other organ systems and tissue types,can be assayed easily. Often, even thebest characterized promoters, althoughclosely reflecting the endogenous gene’sexpression, do not precisely mimic theexact temporal and spatial patterns.Thus, the initial analyses need to be car-ried out in a rigorous fashion on multi-ple lines to determine the effects of copynumber and surrounding chromosomalenvironment on the transgene’s expres-sion. It is, however, difficult, if not im-possible, to gauge the absolute strengthof the promoter accurately solely on thebasis of reporter gene expression. Forexample, if the ultimate purpose of thetransgenic construct is to express a pro-tein whose physiological effects aremanifested only at high concentration(for example, replacement of one of thecomponents of the sarcomere), then thesensitivity of reporter gene detectionmight result in a significant miscalcula-tion of the promoter’s strength. The ac-tual level of transcription might not berobust enough to effect the stoichiomet-ric amounts of protein that are actuallyneeded to modulate the endogenous pro-tein’s concentration.
Promoters derived from genes that areexpressed at high levels specifically inthe heart, such as the MLC2V(Fuller andChien 1994) and the a- and 13-MyHCs,have proved useful (Milano et al. 1994b).For example, the murine MyHC promot-ers have now been extensively character-ized and completely sequenced (Figure1) and have been used to produce ap-proximately 200 separate transgeniclines that express a number of abundantcardiac or muscle proteins. In themouse, the a-MyHC gene is expressedcontinuously in the atria throughout de-velopment, whereas the &MyHC tran-scripts are largely restricted to the ven-tricle during gestation (Figure 1). A few
Figure 1. Thebasicsof transgenedesign.Shownaretheimportantcomponentsandpossiblechoicesin transgenedesignfor cardiacoverexpression.Otherpromotersareavailable(seethetext); shown are the myosin heavychain promoters,both of which havebeen completelysequenced(J.GuliclsandJ.Robbinsunpublisheddata;wMyHC:Genbank#U71441,~-MyHC:#U86076).Thetranscriptionalpatternsof bothpromotersinthedevelopingembryo and in theadult heart are shown above and below the respective constructs. Each promoter was linked tothe reporter gene, ~-galactosidase (M. Colbert and J. Robbins unpublished data), and multipletransgenic lines were generated (Palermo et al. 1996). Transgeneexpression is indicated by thedark blue. Note that the a-MyHC promoter drives expression only in the developing atria,whereas the (3-MyHCsequences drive restricted ventricular expression during gestation. Afterbirth, the a-MyHC promoter drives high levels of expression in all cardiac compartments,whereas the ~-MyHC sequences are significantly downregulated. The exons (filledboxes) andintrons (lines) are indicated in the promoters, the gene, and minigene. Note that in theminigene, some of the exons (redand green boxes) are prespliced in order to reduce the overallsize of the insert. Thus, although the overall splicing patterns (as indicated) will differ betweenthe three types of constructs, the coding sequences should be identical. Either the gene,minigene, or cDNA is linked directly to the promoter of choice. As drawn in this figure, eitherof the three should result in the identical mature transcript. The preferred Kozak sequence isshown, and the methioninecodon is boldface and underscored. The polyadenylation signalsequence and 3’-UTR are shown in gray.A, atrium; v,ventricle; ao, aorta; pa, pulmonary artery;postbirth, 7- to 10-day neonatal hearts
days before birth, &MyHC is shut downand a-MyHC is upregulated in the ven-tricle, where it persists throughout de-velopment (Ng et al. 1991). (3-A4yHCcon-tinues to be expressed in the slowskeletal muscle fiber types in the adultand, so, is not the promoter of choice ifcardiac-specific expression is desired inthe adult. Both promoter constructs usean exon–intron structure that gives riseto the MyHCs’ 5’-untranslated regions(UTRS). The presence of introns in theprima~ transcript apparently enhancesthe efficiency of nuclear processing andis thought to lead to higher steady statelevels of the transgene’s transcript in thecytoplasm (Brinster et al. 1988, Clark etal. 1993), although extensive data on theexact processes responsible are lacking.
Generally, inclusion of at least somesequence 5’ to the AUG translationalstart site is necessary for high levels of
the transgene’s product. The 5’ terminiof eucaryotic mRNA contain a methy-lated cap that protects the end of themessage from exonuclease activity. Italso functions as a ribosomal recogni-tion site for the initial interaction of themessage with the translational appara-tus. The function of the 5’-UTRS of mostmRNAs is not well understood, althoughin some isolated cases, the sequence canhave dramatic effects on both the mes-senger’s ability to be translated and itseffective cytoplasmic half-life (Hess andDuncan 1996, Kakinuma et al. 1996).Empirically, we have noted that for bothMyHC promoters, the resultant process-ing of the introns and the presence of themyosin 5‘-UTR (which is part of anmRNA that is stable and present at veryhigh steady state levels in the cytoplasm)do not destabilize the transgenic tran-script. If transgene expression in both
the adult atria and ventricles is desired,the a-MyHC promoter is appropriate(Palermo et al. 1995). If compartment-specific expression is desired in theadult, for the ventricle the transcrip-tional apparatus of choice would be theMLC2V promoter (Hunter et al. 1995),whereas the atrial natriuretic factor pro-moter could be used to drive transgeneexpression in the atrium (Field 1988).Each of these promoters has been usedeffectively to drive transgene expressionof a biologically active protein.
● The Transgene
The components that should be consid-ered in designing a transgene’s body,that is, the sequences that will be linkedto the ,promoter and subsequently tran-scribed are summarized in Figure 1. Thegeneral 5’-UTR was discussed earlier.Af-ter the ribosome and associated factorsrecognize the methylated cap, the struc-ture migrates down the message until itencounters an AUG initiator codon. Byitself, this triplet is not enough to pro-mote efficient translational initiation.Kozak [for reviews, see Kozak (1994)and (1996)] has shown that the nucle-otides immediately surrounding theAUG (methionine) initiation codon playa particularly important role in the up-stream untranslated region in that theydetermine the efficiency with which aproductive translational initiation com-plex transits the triplet. The activity andimportance of the “Kozak sequence”(Figure 1) on translational initiation canbe quite significant, and it is worthwhile,if the endogenous transgene does nothave a strong Kozak sequence, to reengi-neer this portion of the transgene in or-der to enhance the levels of translationalinitiation over those achieved by com-peting mRNAs. If any upstream se-quence derived from the endogenousmessage is to be included, it should beexamined carefully for ATG codons, asthese could well lead to precocious ini-tiation with a resultant mutated protein.
The body of the transgene encom-passes those nucleotides that encode thefunctional, or perhaps nonfunctional,polypeptide whose expression in the car-diac compartment is the ultimate goal ofthe experiment. A basic decision to bemade is whether to attempt to expressthe entire gene, a minigene, in which a
number of introns have been removed inorder to enhance ease of manipulation,or a complementary DNA (cDNA) thatcontains only those sequences found inthe mature mRNA (Figure 1). In our ex-perience, there is little or no advantagein choosing to express the gene or mini-gene versus the cDNA. If, however, thepromoters being used are intronless, it isprobably productive to engineer a “ge-neric intron” into the construct’s body,along with a 40–150 base s’-UTR. Use ofthe MyHC promoters that contain in-trons and produce 5’-UTRS lead to highlevels of transcription and translation ofthe transgenic cDNAs (Gunteskihamblinet al. 1996, Knotts et al. 1996). Indeed,the use of a gene or minigene may offersignificant disadvantages. To the splic-ing apparatus, all 5’ splicing sites, aswell as 3‘ splicing sites, look very similar,and in principle, any proximal splice sitecan react with any distal site. In practice,in the normal cell this does not happen(or at least, not often enough to causesignificant problems) and the correctsplicing events predominate. The factorsthat determine choice, however, are notwell understood. It is thought that RNAcontext and secondary or tertiary con-formations within the vicinity of thesplice junctions help determine relativeaccessibility. In any case, by making aminigene, or by overexpressing largeamounts of a primary transcript that theprocessing apparatus never has seen, ab-errant splicing events may well occurand lead to physiologically relevant ac-cumulations of mutant mRNAs (Davis-son et al. 1996).
Normally, these problems can beavoided by using the appropriate cDNAconstruct, which has been obtaiaed ei-ther by conventional cloning techniquesor, more and more frequently, by directamplification of the mRNA with thepolymerase chain reaction (PCR). Thelatter technique has significant advan-tages: it is fast (a couple of days) and the5’ and 3’ ends of the construct can beconveniently engineered for future clon-ing steps with the use of appropriateprimers. It is, however, absolutely essen-tial to sequence the entire clone that willbe placed into the promoter vector, asthe thermostable polymerases do havesignificant error frequencies. A numberof companies have now circumventedthis limitation by creating bioengineeredpolymerases that incorporate a proof-
reading activity (for example, Strat-agene’s Pfiu,Boehringer-Mannheim’s Ex-tend HF). In our experience, theseenzymes have undetectable error rates,and their use is strongly recommended.Sequencing of the initial clone is stillessential, but with the increasing use ofautomated sequencers, this does notpresent a particularly significant burdenon the individual laboratory Any subse-quent cloning steps, including the place-ment of the clone into a suitable pro-moter, also necessitate sequencing of thecloning junctions and surroundingnucleotides to ensure that no rearrange-ments or single base deletions have in-advertently occurred.
Translation of the transgene termi-nates with one of the normal termina-tion codons (UAA, UAG, or UGA); thesemust be incorporated into the body ofthe gene or cDNA. Most eucaryotic mR-NAs also contain nucleotides after thetermination codon as well as the poly-adenylation signal, 5’-AAUAAA-3’. Thefunction(s) of these 3’ untranslated re-gions (3’-UT’S) and the polyA tail (-200As) are generally not well understood,although in some specific cases, they actto modulate message stability (Amara etal. 1996, Yeilding et al. 1996). A priori itis difficult to know whether to use theendogenous mRNAs 3’-UTR or a se-quence from a different transcriptwhose mRNA is stable. Our laboratoryroutinely uses approximately 20–40bases of the endogenous mRNAs 3’-UTRand links this to a heterologous se-quence that encodes a polyadenylationsignal. We have found that the polyade-nylation signals derived from either theSV40 T antigen or human growth hor-mone genes both work well and do notappear to confer a negative selective dis-advantage on transgenic transcript accu-mulation. As a cautiona~ measure, if aheterologous 3’-end is engineered intoour constructs, we also include addi-tional termination codons in all threeframes before the polyadenylation signalin order to ensure that read-through ofthe protein does not occur. The authorbelieves, however, that ignorance of thebiological function of the 3’ end of amRNA can be a serious detriment totransgene design. The 3’-UTR of specificmRNAs can have functional conse-quences that are quite unexpected (Ras-tinejad et al. 1993). For example, it ispossible that a 3’-UTR could function to
target the message to the appropriateparts of the cytoplasm for coassembly ofnascent protein into macromolecularstructures. If this were the case, inclu-sion of a heterologous sequence, or in-clusion of only a partial 3’-uTR, couldlead to incorrect targeting and perhapsdegradation of the transgenic transcript.While speculative, these considerationsare worth reviewing, particularly if theexperiment ultimately fails to result insignificant accumulation of the trans-genic polypeptide.
The detection of a transgenictranscriptis relativelystraightforward, as inclusionof the heterologous 5’ or 3’ sequencesusu-ally allows one to design oligonucleotidesthatare capable of distinguishingbetweenthe transgenic and endogenous tran-scripts.Theuse of a cDNA from a differentspecies is relativelycommon in the litera-ture and also allows the design of tran-script specific probes. Cross-species ex-pression, however, is not a favoredapproach in our laboratory. Very often,thereare sequence differences-the signif-icance of which, though often neglected,isnot known. Therefore, one can never besurewhether the alteredphenotype is dueonly to the overexpression of the proteinand the particular mutation that is beingstudied or is simply a result of the pres-ence of the altered,albeitvery similar,het-erologous protein isoform. In order to pre-vent yet another confounding variable inwhat will be a complex series of eventsinunravelingthe eventualphenotype in theanimals,the use of the mouse sequence asstarting material, even if it needs to becloned, is highly recommended.
Detection of the transgenic protein isoften not as easy and is highly depen-dent upon the particular experiment. Ifthe expression is ectopic, that is, the pro-tein isoform is not usually found in theventricle or atria, one can usually detectthe transgenic product directly, either bygel electrophoresis or by Western blots ifa suitable antibody is available. If ex-pression of a subtly mutated proteinwhose wild-type form is abundantly ex-pressed in the heart is carried out, directdetection may be difficult or impossibleand a phenotypic screening for trans-gene expression must be devised prior tocarrying out the experiment. one canoften design the construct so that a trun-cated or extended form of the proteinresults, and this species can be separatedfrom the wild-type isoform on polyacry-
lamide gels. Alternatively, one can lookfor increases in the absolute or relativeamounts of the protein species as evi-dence for transgene expression.
An alternative approach, if there areno other means of detection, is to incor-porate an epitope tag into the body ofthe translated transgenic construct. Anumber of defined epitope tags such asc-myc (EQLISEEDL), FLAG (DYKD-DDDDK), or vesicular stomatitis virusglycoprotein or VSV (YTDIEMNRLGK)are available whose presence in the re-sultant protein can be detected easilywith the use of commercially available,highly sensitive antibodies. It is impor-tant when engineering these peptide se-quences into the construct that the pref-erential codon usage of the mousetranslational apparatus be taken into ac-count. Most sequence analysis programshave the capability to determine codonusage frequencies, and this should bedone for the particular gene/cDNA un-der study. Normally the tags are placedat the N or C terminus in order to min-imize the possibility of interfering withthe protein’s function. The advantages ofthe epitope tagged approach are signifi-cant: one can easily detect the transgenicproduct, quantify the steady-state levelof the protein, determine whether it ispresent in the correct intracellular loca-tion, and purify it. The disadvantage ofepitope tagging is that considering therudimentary state of effective proteinmodeling, it is often impossible to deter-mine whether incorporation will havesubtle or major effects on protein func-tion. Although a major effect (degrada-tion, loss of enzymatic activity, and soforth) could be detected easily, subtlechanges in the protein’s function orstructure could be easily missed yet re-sult in a discernible phenotype thatwould then be ascribed to either the pro-tein’s overexpression or presence of themutation under study. When epitopetags are used, it may be productive toconstruct, in parallel, a transgene thatdoes not contain the epitope. Both setsof founders are subsequently generated,bred, and studied so that the effects oftransgenic expression with and withoutthe epitope tag can be rigorously deter-mined. Although not entirely satisfac-tory such an approach gives more com-plete data sets.
● Animal Productionand InitialAnalyses
A perceived disadvantage of the generaltransgenic approach is the length of timerequired until an initial set of animals isavailable for analysis. The time line, al-though significant, is certainly within anormal time frame for a molecular bio-logical or physiological study. Gestation,after injection of the fertilized eggs withthe construct DNA and implantationinto a pseudopregnant female, is ap-proximately 3 weeks. Different facilitiesprefer different strains of mice, theirchoices being based on robustness andfecundity of the strain, the size of thepronucleus, and the litter sizes, amongother considerations. We have found,however, that a critical parameter is thestability of transgene expression as it istransmitted vertically through multiplegenerations. In our experience, use ofthe FVB/N strain results in invarianttransgene expression through at least 10generations. In our facility, approxi-mately 100 fertilized embryos are in-jected per construct, resulting in 30-60live births. The offspring can bescreened within 2 weeks, and thefounders (usually 5–15) are identified ei-ther by PCR or Southern analyses.Founders, termed FOS,are not usuallyanalyzed further,because of the possibil-ity that the transgene may not be presentin all cell types (mosaicist). Thefounder mice reach sexual maturitywithin another 5 weeks and are immedi-ately outbred to nontransgenic mates inorder to determine whether the trans-gene is carried through the germline.Positive offspring, the Fls, can be iden-tified within 2 weeks and are either al-lowed to reach sexual maturity in orderto breed up the colony or, depending onthe experiment, are analyzed immedi-ately for transcript abundance, proteinaccumulation, and transgene copy num-ber. Thus, one has identified founders5-6 weeks after submission of the DNAfor injection and can begin the initialanalyses on the Fls within 16 weeks.
The production of multiple lines oftransgenic founders is often imperativefor the ultimatesuccess of the experiment.Dependence upon data generated fromone or two lines is problematic owing toposition dependent effects or insertionalmutagenesis. Insertion of the transgenicDNA into the chromosomal DNA, al-
though usuallyoccurring ata singlesite,isnot a benign process and can leadto trans-locations or massive deletions. Recapitu-lation of the observed phenotype in mul-tiple lines is necessary to rule out thisartifact. Similarly, it is prudent to avoidbreeding theresultantanimalsto homozy-gosity.This minimizes any insertionalde-fects that are present, as they are oftenrecessive, and the presence of one wild-type allele is sufficient to preserve thenormal phenotype. ,knecdotally,it is com-monly assumed that insertionalmutagen-esis is a relatively rare event, occurringonly in about 100/0of the animals,but thisis probably an underestimation resultingfrom the superficial nature of most pre-liminary screenings. In a set of experi-ments carried out over a 6-month periodat the University of Cincinnati’s Trans-genic Facility randomly chosen con-structs were bred to homozygosity andrigorously screened for obvious abnor-malities in all organ systems. The inci-dence of detection was 3 times thatnumber (J. Neumann personal communi-cation). Therefore, the possibility that aphenotype is due to an insertional mu-tagenic event cannot be discounted with-out the analysis of multiple, indepen-dently generatedlines.
Normally, a first set of analyses is car-ried out at the transcriptional and pro-tein levels on 5–7 founder lines. If fewerfounders result from the initial set ofinjections, the mice are subjected to atleast these prelimina~ analyses beforeundertaking another round of injec-tions. Probably 3 is the minimum num-ber of founders with which the experi-ment can be carried forward; anythingless and the risk of insertional mutantsor pc)sition dependent effects seriouslycompromises the experimental para-digm. An additional advantage in ana-lyzing multiple founders is that often adose-response curve can be established.Almost always, different lines expressthe transgene at different levels: for theMyHC promoters, expression is partiallya function of copy number, with highercopy numbers showing higher levels ofsteady-state transgenic transcript accu-mulation. Thus, with a single construct,one can determine the phenotypic ef-fects of gradually increasing the dose ofthe transgenic product.
If few or no founders are detected, thecauses must be carefully considered.Technical considerations include a poor
preparation of DNA or a “bad day” at theinjection facility. The successful genera-tion of founders is quite sensitive to thequality of the nucleic acid used. Nor-mally, the DNA is freed of all vector se-quences, exhaustively purified, and thendialyzed before injection. Many facilitiesoffer to prepare and purify the DNA forinjection for a fee. The novice is stronglyencouraged to take advantage of this ser-vice, as the generation of DNA suitablefor injection can be technically demand-ing for the less experienced molecularbiologist. Using the facility’s servicesmeans one less experimental variableand can, if no founders are born, resultin another imundof injections at mini-mal or no cost to the investigator. Thelack of founders can also be an indica-tion of transgene lethality, which canonly be tested by carrying out anotherseries of injections, timing the pregnan-cies, and genotyping the embryos beforethe stage of gestation at which they ex-pire and are reabsorbed. For proteinsthat are involved in cardiac function andcause lethality, this usually occurs be-tween embryonic days 11.5 and 13. Iflittle or no transgenic mRNA can befound, or if significant levels of tran-script are made but no protein can bedetected, troubleshooting can be alengthy and difficult process.
If a reasonable number of foundersare generated but none contain signifi-cant levels of the transgenic transcript, itis likely that there is either a basic flawin the construct or that a processing ar-tifact is leading to rapid turnover of anaberrant message. There are many pos-sible control points between mRNA ac-cumulation and the production of bio-logically active protein in the cytoplasm.One of the more likely points at whichthe process can go awry is during pro-cessing of the primary transcript. Asnoted earlier here, exon–intron splicingis not well understood, and cryptic splicejunctions can be inadvertently made andrecognized by the spliceosomes, result-ing in aberrant splicing even in a cDNA.This has occurred in a transgenic con-struct made with the MLC2a cDNA (J.Gulick and J. Robbins unpublisheddata). Although in some cases the result-ant mRNA’s size is altered, in other casesthe mutated mRNA cannot be discernedfrom the correctly sized message. If noprotein is made, it may be necessary toexamine the transgenic transcript’s se-
quence directly, using a combination ofreverse transcriptase and PCR. If an ab-errant splice junction is identified, theoriginal construct can be altered in sucha manner that the splice junction is abol-ished.
Levels of the transgenic protein mayalso be highly dependent on the specificprotein being expressed, as well on asthe biology of the system. Therefore, theinvestigator must carefully consider theposttranslational processes (protein sta-bility, trafficking, posttranslational pro-cessing or modification) that might im-pact on protein accumulation orbiological activity. For example, in ex-periments in which the MLCS are over-expressed in the heart, the cardiomyo-cyte rigorously controls the absoluteamounts of the total light chain isoformsin each cardiac compartment, presum-ably by increasing degradation rates.Thus, even though the total MLC2Vtran-script is present in 10-fold excess in thetransgenic ventricle, no significant in-crease in the cognate protein occurs(Palermo et al. 1996). It should be notedthat this may represent a specializedcase: the protein is soluble and the sto-ichiometry of the sarcomeric proteinsmust be conserved during assembly ofthe contractile apparatus. If protein lev-els are disappointingly low or nearly un-detectable, this may indicate that thetransgenic protein is inefficiently incor-porated into the appropriate macromo-lecular system (Gulick et al. 1997). Pos-sibly, higher levels are lethal and so micein which expression was robust nevercome to term. A low number of foundersor small litter sizes would support thisinterpretation, and the hypothesis canbe tested by breeding offspring from in-dependent lines to one another in hopesof increasing gene dosage in the doubleheterozygotes and noting reabsorptionplaques during a timed pregnancy.
The transcript and protein analysesare merely the starting points for analyz-ing the animals. The subsequent analy-ses will depend upon the particular biol-ogy of the experiment. One of the majoradvantages of transgenic remodelingversus in vitro assays is the potential forstudying the biological consequences atthe whole organ level over the animal’slifetime (senescence sets in at approxi-mately 11 months, although mice canlive as long as 2 years). Great strideshave been made in applying standard
methodologies for measuring heartfunction to the mouse system, and thelimitations inherent in the small rodent’sheart have been or rapidly are beingovercome (Becker et al. 1996, Chien1996). Another significant advantage inusing transgenics is that the animals canbe bred onto different genetic back-grounds or with different genetically en-gineered mouse strains. Thus, the conse-quences of multifactorial changes in thecardiac protein complement can bestudied during cardiac differentiationand development, as well as in the intactadult animals. A spectrum of molecular,biochemical, cytological, pathologic,histologic, and physiologic analyses canbe subsequently carried out in order toilluminate the consequences of the al-tered protein profiles on normal and ab-normal cardiac function. The animalscan also serve as an invaluable “factory”for the synthesis of modified or mutatedproteins suitable for ex vivo and in vitrobiochemical studies. The value of allthese reagents will be enhanced signifi-cantly as they are shared freely amongmembers in the fields of cardiovascularmedicine and research.
● Acknowledgments
Work carried out in the author’s labora-tory is supported in part by grants fromthe National Institutes of Health. Thecontributions of James Gulick, ArurtSubramaniam, Hansjorg Rindt, andStephanie Knotts to the transgenic workin the Robbins laboratory are particu-larly appreciated.
References
Amara FM, Sun J, Wright JA: 1996. Defining anovel cis-element in the 3’-untranslated re-gion of mammalian ribonucleotide reductasecomponent R2 mRNA. Cis-trans-interactionsand message stability. J Biol Chem 271:20,126-20,131.
AnversaP, Capasso JM, OlivettiG, et al.: 1992.Cellular basis of ventricular remodeling inhypertensive cardiomyopathy. Am J Hypert5:758-770.
AraiM, AlpertNR, MacLennan DH, etal.: 1993.Alterations in sarcoplasmic reticulum geneexpressionin human heart failure:a possiblemechanism for alterationsin systolic and di-astolic properties of the failing myocardium.Circ Res 72:463469.
Barany M: 1967. ATPase activity of myosin is osin light chains in the mammalian heart.correlated with the speed of muscle shorten-ing. Science 231:597+00.
Biben C, Hadchouel J, TajbakhshS, et al.: 1996.Developmentaland tissue-specificregulationof the murine cardiac actin gene in vivo de-pends on distinct skeletaland cardiac mus-cle-specific enhancer elements in addition tothe proximal promoter. Dev Biol 173:200–212.
Blaufarb IS, Sonnenblick EH: 1996.The renin–angiotensin system in leftventricularremod-eling. Am J Cardiol 77:8C–16C.
Boluyt MO, O’NeillL, MeredithAL, et al.: 1994.Alterationsin cardiac gene expressionduringthe transition from stable hypertrophy toheart failure: marked upregulation of genesencoding extraceflular matrix components.Circ Res 75:23-32.
Brinster RL, Allen JM, Behringer RR, et aL:1988. Introns increase transcriptional effi-ciency in transgenicmice. Proc NatlAcad SciUSA 85:836-840.
Buttrick PM, Kaplan ML, Kitsis RN, et aL:1993. Distinct behavior of cardiac myosinheavy chain gene constructs in vivo: discor-dance with in vitro results.Circ Res 72:1211-1217.
Chien KR: 1996. Genes and physiology: molec-ular physiology in geneticallyengineeredan-imals. J Clin Invest 97:901–909.
ClarkAJ,Archibald AL, McClenaghan M, et al.:1993. Enhancing the efficiency of transgeneexpression. Philos Trans R Soc Lend B BiolSci 339:225-232.
Davisson RJ+Nuutinen N, Coleman ST, et aL:1996. Inappropriate splicing of a chimericgene containing a large internalexon resultsin exon skipping in transgenicmice. NucleicAcids Res 24:4023-4028.
Field LJ: 1988. Atrial natriureticfactor-SV40 Tantigen transgenesproduce tumors and car-diac arrhythmiasin mice. Science 239:1029-1033.
Field LJ: 1993. Transgenicmice in cardiovascu-lar research.Ann Rev Physiol 55:97-1 14.
FigueredoVM, Camacho SA: 1995.Basic mech-anisms of myocardiaf dysfunction: cellularpathophysiology of heart failure. Curr OpinCardiol 10:246-252.
Francis GS, McDonald K, Chu C, et al.: 1995.Pathophysiologic aspects of end-stage heartfailure. AmJCardiol75:11A-16A.
FullerSJ, Chien KR: 1994. Genetic engineeringof cardiac muscle cells: in vitro and in vivo.Genet Eng 16:17-31.
Gulick J, Hewett TE, KfevitskyR, et al.: 1997.Transgenicremodeling of the regulatorymy-
Circ Res 80:664–665.Gunteskihamblin AM, Song GJ, Wafsh RA, et
al.: 1996. Annexinvi overexpressiontargetedto heart alters cardiomyocyte function intransgenic mice. Am J Physiol 39:H1091–H11OO.
HunterJJ,TanakaN, Rockman HA,et al.: 1995.Ventricular expression of a MLC-2v-ras fu-sion gene induces cardiac hypertrophy andselective diastolic dysfunction in transgenicmice. J Biol Chem 270:23,173–23,178.
Kakinuma A, Chazenbalk G, Filetti S, et al.:1996.Both the 5’ and 3’ noncoding regionsofthe thyrotropin receptor messenger ribo-nucleic acid influence the level of receptorprotein expression in transected mamma-lian cells. Endocrinology 137:2664-2669.
Knotts S, Sanchez A, Rindt H, et al.: 1996.Developmentafmodulation of a beta myosinheavy chain promoter-driventransgene.DevDyn 206:182-192.
Kozak M: 1994. Determinantsof translationalfidelity and efficiency in vertebratemRNAs.Biochimie 76:815-821.
Kozak M: 1996. InterpretingcDNA sequences:some insights from studies on translation.Mamm Genome 7:563-574,
Liu P, Penninger J, AitkenK, et af.: 1995. Therole of transgenicknockout models in defin-ing the pathogenesis of viral heart disease.Eur Heart J 16(Suppl 0):25-27.
Milano CA, Aflen LF, Rockman HA, et al.:1994a. Enhanced myocardial function intransgenic mice overexpressing the beta2-adrenergicreceptor.Science 264:582-586.
Mikmo CA, Dolber PC, Rockman HA, et aL:1994b. Myocardiaf expression of a constitu-
Molkentin JD, Markham BE: 1994. An M-CATbinding factor and an RSRF-related A-richbinding factor positively regulate expressionof the alpha-cardiac myosin heavy-chaingene in vivo. Mol Cell Biol 14:5056-5065.
Moranc) M, Zacharzowski U, Maier M, et al.:1996. Regulation of human heart contractil-ityby essentiafmyosin lightchain isoforms. JClin Invest98:467-473.
Ng WA,Grupp IL, Subramaniam A, et al.: 1991.Cardiac myosin heavy chain mRNA expres-sion and myocardial function in the mouseheart. Circ Res 68:1742-1750,
Palermo J, GufickJ, Ng W, et af.: 1995.Remod-eling the mammalian heart using transgen-esis. Cell Mol Biol Res 41:501–509.
Palermo J, Gulick J, Colbert M, et al.: 1996.Transgenicremodeling of the contractile ap-paratus in the mammalian heart. Circ Res78:504-509.
PayneRM, Johnson MC, GrantJW,et al.: 1995.Toward a molecular understanding of con-genitalheartdisease.Circulation91:494-504.
Rastinejad F, Conboy MJ, Rando TA, et al.:1993.Tumor suppressionby RNA from the 3’untranslated region of alpha-tropomyosin.Cell 75:1107-1 117.
Rindt H, Subramaniam A, Robbins J: 1995. Anin vivo anafysisof transcriptionalelementsinthe mouse alpha-myosin heavy chain genepromoter. TransgenicRes 4:397+05.
Rubin EM, Smith DJ: 1994. Atherosclerosis inmice: getting to the heart of a polygenic dis-order;TrendsGenet 10:199–203.
Yeilding NM, Rehman MT, Lee WM: 1996.Identification of sequences in c-myc mRNAthat regulate its steady-statelevels. Mol CellBiol 16:3511–3522. TCM
REPRINTSReprints of articles in TCM are available (minimum order: 100).
