R ev is t a d a S o c ie d a d e B ra s ile ira d e M e d i c in a T ro p ica l 2 9 ( 3 ) : 2 8 7 - 2 8 9 , m a i- j u n , 1 9 9 6 .
CO M UN ICA ÇÃ O
TH E SECO N D BURITI W O RK SH O P O N
TRA N SFECTIO N O F PARASITES
Augusto Simões-Barbosa, N ancy R. Sturm and A ntônio R.L Teixeira
The d ev elo p m ent o f new techno lo g ies fo r intro d ucing exo g eno u s DNA into p arasites has a llo w e d th e stu d y o f p r o te in f u n c tio n , m e c h an ism s o f g e n e e x p re ssio n and its reg ulatio n, drug resistance and d esig ning o f new p o tential chem o therap eu tic targets. The u se o f these m o d ern techno lo g ies w as the m ain o b jectiv e o f the Seco nd Buriti W o rksho p o n Transfectio n o f DNA into Parasites, that to o k p lace at the Labo rato ry fo r Multidisciplinary Research o n Chagas D isease, Faculty o f H ealth Sc iences o f the Univ ersity o f Brasilia, fro m the 1st to 15 o f Ju ly , 1995. T h e W o rksh o p includ ed thirteen inv estig ato rs fro m v ario us Braz ilian and fo reig n research institutio ns, and an eq u al nu m b er o f stu d ents that w ere selected fro m the cand id ate ap p licants fro m São Paulo , Minas Gerais, Go iás and Pernam buco , and the Fed eral District.
The theo retical asp ects o f the W o rksho p w ere intro d uced b y N ancy R. Sturm (Dep artm ent o f M icro b io lo g y and Im m u no lo g y , UCLA Sc h o o l o f M ed icine), w ho d iscu ssed the d ifferent m etho d s o f transfectio n alo ng w ith req u irem ents fo r elec tro p o ratio n and the v ario us exp erim ental co nd itio ns d efined in the W o rk s h o p p r o to c o ls . T h e s p e a k e r a ls o d iscussed so m e p eculiar b io lo g ical features o f kineto p lastid p ro to z o a such as m iniexo n g ene array, p o ly ad eny latio n and c o d o n usag e, and p o ly cistro nic seq u ences and m aturatio n o f mRNA.
Jo h n M. Kelly (Lo nd o n Scho o l o f Tro p ical M ed icine) rep o rted o n the v ecto r he d esig ned fo r transfectio n o f T ry p a no s o m a c ru z i and L e is h m a n i a sp. This v ecto r is b ased o n the p lasm id p Blu escrip t and co ntains tw o co p ies
Lab oratório M ultidisciplinar de Pesq uisa em D oen ça de Chagas. Faculd ade de C iências da Saúde, U niversidade de Brasília, Brasília, D F e D ep artm ent o f M icrob iology and Im m unology, UCLA School o f M edicine, Los A ngeles, CA .
A d d r e s s t o : D r. A n to n io R .L. T e ixe ira, L ab o rató rio
M ultid iscip linar d e Pesq uisa em D o en ça de C hagas, Faculd ade de C iências da Saúde, U niversidade de Brasília. C aixa Postal 0 4 - 6 8 5 ,7 0 9 1 9 - 9 7 0 Brasília, DF, Brasil. Fax: (0 6 1 ) 273- 4645.
Receb id o p ara p ub licação em 2 8 / 0 2 / 9 6 .
o f the GA PDH g ene, and the neo m y cin p ho sp ho transferase g ene as a selected marker. The v ecto r characteristics includ e ep iso m al rep licatio n and transfo rm atio n o f p arasites resulting in exp ressio n (1% to 10% o f to tal cellular p ro tein) o f reco m b inant p ro teins. The transfected p heno ty p e c an b e m aintained thro ug h the entire T. c ru z i life cy cle in the p ro lo ng ed ab sence o f drug selectio n. The p TEX v ecto r has b e e n u sed to stud y the functio nal asp ects o f cruzipain and tryp ano thio ne reductase, p utativ e targets fo r chem o therap y 3 5 7.
D av id A . C a m p b e ll ( D e p a r tm e n t o f M icro bio lo g y and Im m uno lo g y , UCLA Scho o l o f M e d i c i n e ) a n d D a v id M . E n g m a n (D ep artm ent o f Patho lo g y , N o rthw estern U n iv ersity Sc h o o l o f M e d ic in e , C h ic ag o ) d iscu ssed sev eral m etho d s fo r analy sing transfected cells using DNA , RNA and p ro tein analy sis. A fter stud y ing sev eral m o lecu lar b io lo g y m etho d o lo g ies, the stud ents co u ld see the im p o rtance o f the p articular b io lo g ical features o f these o rg anism s. Fo r exam p le, try p ano so m atid p ro tein co d ing g en es are exp ressed as o p ero ns w ith 5’ cap p ing , tans- sp licing m echanism , and 3 ’ p o ly ad eny latio n. The d iscussio n led to the m etho d s o f ad d ing an ep ito p e tag at the carbo xy l-term inal end o f the reco m b inant p ro tein and d eterm ining its subcellular lo calizatio n.
Elibio Rech (Cenarg en, Brasília, BR) rep o rted o n the bio listic p rincip les o f transfectio n, e x e m p l i f i e d b y th e “ g e n e g u n ” . T h e m icro bo m bard m ent p ro cess uses helium g as at hig h p ressure and D N A -co ated p articles. The p articles co ated w ith DNA reach 1500km / h and reach all parts o f the cell, includ ing the o rg anelles. This p ro ced ure is v ery usefull in p lant cells since their c ell w alls d o no t p erm it the entrance o f exo g eno u s DNA b y o ther m etho d o f transfectio n (e.g ., lip o fectio n o r p recip itatio n w ith calcium p ho sp hate). The p o tential fo r im m uniz atio n using the g ene gun is d em o nstrated b y the su ccessfu ll v accinatio n ag ainst fo o t and m o uth d isease in cattle7.
A ng ela Cruz (Faculd ad e d e M ed icina d e Ribeirão Preto , SP) sp o ke ab o u t g ene targetting
C o m u n ic a ç a o . S im õ e s - B a rb o s a A , S t u rm NR, T eix eira A R L. T he S e c o n d B u ri t i W o rks ho p o n T ra n s fe c t io n o f P a ra s it es . R ev is t a d a S o c ie d a d e B ra s ile ira d e M e d i c in a T ro p ica l 2 9 : 2 8 7 - 2 8 9 , m a i- j u n , 1 9 9 6 .
and g eno m e analy sis. Using electro p o ratio n tec h n iq u es she o b tain ed integ ratio n and r e p la c e m e n t o f th e D H FR -T S g e n e o f L e i s h m a n i a m ajo r. Su ccess w as o b tained using a no n-v im lent cell line but, w hen she attem peted d o u ble kn o c k o ut o f the g enes o f a v irulent cell line, the p arasite b ecam e either tetrap lo id o r aneo p lo id to av o id lo ss o f the g ene. A m o ng the transfec tants, sh e saw ho m o lo g o us reco m binatio n, rando m integratio ns and multiple reco m binatio n. W ith the aid o f a co sm id library tho usand s o f reco m b inants w ere analy z ed by an im p ro v ed m etho d o lo g y 12 6.
Lucille Flo eter-W inter (D ep artam ento d e Parasito lo g ia, ICB, USP) sp o ke o n the use o f transfectio n to stud y g ene p ro m o ters. The analy sis o f transient transfectio n o f T. c m z i and L e is h m a n i a sp . w ith d ifferent co nstructs co ntaining seq u ential d eletio ns up stream fro m the RNA p o l I g ene, she id entified seq u ences co ntaining p ro m o ters o f g ene exp ressio n. The p ro m o ter reg io ns are v ery sp ecific, since they d id no t fu nctio n fo r C hrit id ia fa s c ic u la t a and in so m e strains o f T. cruz i.
Bianca Z ing ales (Instituto d e Q uím ica, USP) sho w ed that sera fro m Chagas heart d isease p atients cro ss-reacted w ith the reco m binant B13 p ro tein fro m T. c ru z i and w ith the m y o sin fro m card iac m uscle cells. O f interest, these p ro teins share an ep ito p e o f 6 o r 7 am ino acid s. Further characteriz atio n o f this ep ito p e is b eing u nd ertaken.
E x p e ri m e n t a l w o rk . Th e se c o n d Buriti W o rksho p co ncentrated o n ho w to d esig n a su c c e ssf u l tran sf e c tio n e x p e rim e n t u sin g elec tro p o ratio n tec h niq u es. Sem inars and p ractical exercises d em o nstrated the step s required fo r electro p o ratio n, such as testing the p aram eters o f the electro p o ratio n m achine, kno w led g e o f the c ell ty p es u sed in the exp erim ents (d rug resistance, gro w th curve, e tc .), and co n firm atio n o f results u sing neg ativ e and p o sitiv e co ntro ls.
Characteriz atio n o f the transfected cells w as u nd ertaken using g eno m ic DNA d ig estio n and hy brid iz atio n b y DNA d o t and so uthern b lo t to d em o nstrate the p resence o f the transfected g en e o f interest and also , the nature o f the transfected DNA (ep iso m al o r integ rated ) and an estim ativ e o f co p y num ber p resent in the cell. RNA analy sis b y no rthern b lo t d etected mRNA w ith sp ecific p ro b e thus sho w ing that the exo g eno u s g ene w as transcribed . The exp erim ents sho w ed ho w exp ressio n o f the
reco m binant p ro tein w as regulated . Further, p ro tein analy sis o f rep o rter g enes sho w ed the v ecto rs used w o rk equally w ell w ith L e is hm a nia t a rent o la e, C. f a s c ic u la t a and T. c m z i . The v e c to rs and th e re p o rte r g e n e s, th e re fo re , c o n ta in th e s ig n a ls th e s e k in e to p la s tid p ro to z o ann p arasites need fo r exp ressio n.
In brief, d uring tw o w eeks instructo rs and stud ents that assisted the W o rksho p d esig ned and p erfo rm ed exp erim ents using transfectio n p ro ced ures. Transfectio n o f p arasites has b een u sed to stud y p ro tein functio n, m echanism s o f g ene exp ressio n and reg ulatio n (e.g ., p ro m o ter seq u ences), and g ene p ro d ucts inv o lv ed in v irulence and drug resistance. The p arasites that w ere electro p o rated and transfected ( I. t a rent o la e, C. f a s c ic u la t a and T. c ru z i) serv ed as to o ls fo r o p tim iz atio n o f th e w o rk in g co nd itio ns. The exp erim ents led the stud ents to carry o n DNA and RNA analy ses. Fo r instance, the stu d ents d em o nstrated the p resence o f the luciferase exo g eno u s g ene in transfected T. c ru z i cells. Further, reco m binant p ro teins w ere sho w n b y W estern b lo t and im m uno cyto chem istry assay s. The reco m binant m H SP70 19E10 and FC B9 9E10 w ere lo caliz ed at the kineto p last and flag ellum , resp ectiv ely , o f transfected T. c ru z i cells, u sing 9E10 ep ito p e tag antibo d y .
The Seco nd Buriti W o rksho p gav e the s tu d e n ts th e o p p o r tu n ity to p e r f o rm electro p o ratio n o f p arasites and ad v anced m o le c u la r b i o l o g i c a l , b i o c h e m ic a l an d im m uno lo g ical techniq u es fo r characteriz atio n o f g ene exp ressio n in transfected cells. The ap p licatio ns o f these tec h n iq u es w ill b e fund am ental in d ev elo p ing new research to o ls to inv estig ate the kineto p lastid p ro to z o an p arasites that cause v ario us end em ic d iseases in the N ew World.
A CK N O W LED G M EN TS
T h e O rg a n iz e rs o f th e Se c o n d B u r iti W o rksho p ackno w led g e the financial sup p o rt fro m PA DCT/ CAPES and FAPDF, and technical assistance fro m Pharm acia Bio tech and Sine d o Brasil.
REFEREN CES
1. Cruz A , Beverly SM. G ene rep lacem ent in parasite protozoa. N ature 3 4 8 :1 7 1 - 1 7 3 ,1 9 9 0 .
2. Cruz A , C oburn CM , Beverly SM. D ouble targeted gene rep lacem ent for creating null m utants.
C o m u n ic a ç ã o . S im õ e s - B a rb o s a A , S t u rm NR, T e ix e ira A R I . T he S e c o n d B u ri t i W o rks ho p o n T ra n s fe ct io n o f P a ra s it es . R eiiista d a S o c ie d a d e B ra s ile ira d e M e d i c in a T ro p ica l 2 9 : 2 8 7 - 2 8 9 , m a i- j u n , 1 9 9 6 .
Proceedings N ational A cadem y Sciences 8 8 :7170-7 1 :7170-7 4 ,1 9 9 1 .
3. Kelly JM , Tom as A M . A n ap proach to functional com plem entation b y introduction o f large DNA f ra g m e n ts i n to T r y p a n o s o m a c r u z i an d L e i s h m a n ia d o n o v a n i using a shuttle vector. M olecular Biochem ical Parasitology 6 5 :5 1 - 6 2 , 1994.
4. Kelly JM , W ard HM , M iles M A, Kendall G. A shuttle v e cto r w h ich f acilitate s th e e xp re ssio n o f transfected genes in T ry p a no s o m a c ru z i and L e is h m a n ia . N ucleic A cids Research 2 0 :3963-3969.
5. LeBow itz JH , C ob urn CM , M cM ahon Prat D, Beverly SM. D evelopm ent o f a stable L e is h m a n ia
expression v ecto r and application to th e study of p arasite surface antigen genes. Pro ceed in g s N ational A cadem y Sciences 8 7 :9 7 3 6 - 9 7 4 0 ,1 9 9 0 .
6. Pasion SG, Brow n GW. Brow n LM , Ray DS. Periodic expression o f nuclear and m itochondrial DNA replication genes during the trypanosom atid cell cycle. Journal Cell Sciences 1 0 7 :3 5 1 5 - 3 5 2 0 , 1994.
7. V ainstein M H, A lves SA, de Lima BD, A ragao JL, R ech EL. Stab le transf ectio n in a flagellate trypanosom atid b y m icroparticle b om b ard m ent. N ucleic A cids Research 2 2 :3 2 6 3 - 3 2 6 4 , 1994.
