Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique

Gene Transfer Technique

Hongbao Ma, Guozhong Chen Michigan State University, East Lansing, MI 48823, USA, hongbao@msu e!u Abstract: This artic e is descri!ing the nine "rinci" e techniques for the gene transfection, #hich are $ (1) i"id%&ediated &ethod' (2) ca ciu&%"hos"hate &ediated' (3) ()*)%de+tran%&ediated' (,) e ectro"oration' (5) !io istics (gene gun)' (-) vira" vectors# $%& 'o"ybrene# $8& "aser trans(ection# $)& gene trans(ection enhance! by e"evate! tem'erature, as the re(erences (or the researchers *ho are intereste! in this (ie"! +,ature an! Science 2--.#3$/&02.13/2 Key words: (N*' gene' technique' transfer agricu"tura" 'otentia" an! me!ica" im'ortance $9am'be"", /)))# U;ogara, 2---# Lorence, 2--4& 8he gene trans(er metho!s norma""y inc"u!e three categories0 / trans(ection by biochemica" metho!s# 2 trans(ection by 'hysica" metho!s# 3 virus1me!iate"y trans!uction 8he gene trans(er resu"ts can be transient an! stab"e trans(ection 3ene thera'y can be !e(ine! as the !e"iberate trans(er o( 4,A (or thera'eutic 'ur'oses Many serious !iseases such as the tragic menta" an! 'hysica" han!ica's cause! by some genetic metabo"ic !isor!ers may be hea"e! by gene trans(er 'rotoco" 3ene trans(er is one o( the 7ey (actors in gene thera'y $Matsui, 2--3&, an! it is one o( the 7ey 'ur'oses o( the c"one $Ma, 2--4& 3ene trans(er can be targete! to somatic $bo!y& or germ $egg an! s'erm& ce""s In somatic gene trans(er the reci'ient6s genome is change!, but the change *i"" not be 'asse! on to the ne5t generation In germ"ine gene trans(er, the 'arents6 egg an! s'erm ce""s are change! *ith the goa" o( 'assing on the changes to their o((s'ring 3erm"ine gene trans(er is not being active"y investigate!, at "east in "arger anima"s an! humans $<or!ignon, 2--3# Umemoto, 2--.& 2 Transient and Stable Transfection 2.1 Transient transfection In transient trans(ection, the trans(ecte! 4,A is not integrate! into host chromosome 4,A is trans(erre! into a reci'ient ce"" in or!er to obtain a

1 Introduction 3ene trans(er is to trans(er a gene (rom one 4,A mo"ecu"e to another 4,A mo"ecu"e 3ene trans(er re'resents a re"ative"y ne* 'ossibi"ity (or the treatment o( rare genetic !isor!ers an! common mu"ti(actoria" !iseases by changing the e5'ression o( a 'erson6s genes $Arat, 2--/& In /)28, 3ri((ith re'orte! that a non'athogenic 'neumoccocus strain cou"! become 'athogenic *hen it *as mi5e! *ith ce""s o( heat17i""e! 'athogenic 'neumoccocus, *hich hinte! that the 'athogenic genetic materia" cou"! be trans(orme! (rom the heat17i""e! 'athogenic 'neumoccocus to the non'athogenic strain $3ri((ith, /)28& 8his is the (irst re'ort (or gene trans(er observation Ho*ever, the trans(orming substance *as not i!enti(ie! in these e5'eriments U' to /)44, Avery et a" !emonstrate! that !eo5yribonuc"eic aci! $4,A& *as the trans(orming substance $Avery, /)44& In /).2, Hershey an! 9hase sho*e! that 4,A *as the on"y materia" trans(erre! !uring bacterio'hage in(ection, *hich suggeste! that the 4,A is the genetic materia" $Hershey, /).2& 8he basic techni:ue (or intro!ucing 4,A into E co"i have ins'ire! 'roce!ures (or the intro!uction o( 4,A into ce""s (rom a *i!e variety o( organisms, inc"u!ing mamma"ian ce""s 3enetic engineering o( (oo! is the science *hich invo"ves !e"iberate mo!i(ication o( the genetic materia" o( '"ants or anima"s Intro!uction o( 4,A into '"ants is o( great

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Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique tem'orary but high "eve" o( e5'ression o( t he target gene. 2.2 Stable transfection Stab"e trans(ection is a"so ca""e! 'ermanent transfection. /0 the sta! e transfection, the transferred (N* is integrated (inserted) into chro&oso&a (N* and the genetics of reci"ient ce s is "er&anent changed. 3 Transfection Methods 3enera""y, there are ) *ays (or gene trans(er0 $/& Li'i!1me!iate! metho!# $2& 9a"cium1'hos'hate me!% iate!# $3& 4EAE1!e5tran1me!iate!# $4& E"ectro'oration# $.& <io"istics# $?& @ira" vectors# $%& Ao"ybrene# $8& La1 ser trans(ection# $)& 3ene trans(ection enhance! by e"e1 vate! tem'erature $Sambroo7, 2--/& 3.1 Lipid-mediated method 8his metho! can be use! (or both transient an! stab"e trans(ection, an! it can be use! (or a!herent ce""s, 'rimary ce"" "ines, an! sus'ension cu"tures B or the fo o#ing "rotoco , the 1i"ofecta&ine 2eagent fro& Invitrogen 9or'oration *i"" be use! Li'o(ectamine Ceagent is a 30/ $*=*& "i'osome (ormu"ation o( the '"ycationic "i'i! 2,31!io"ey"o5y1,1 +2$s'erminecar!o5% i!o&ethy"21,,,1!imethy"1/1'ro'anaminium tri("uoro% acetate $4DSAA& an! the neutra" "i'i! !io"eoy" 'hos'hati!y"ethano"amine $4DAE& in membrane1 (i"tere! *ater $9ata"ogue ,umber /8324, Invitrogen 9or'oration, 9ar"sba!, 9a"i(ornia, USA& $Ha*"ey1 ,e"son, /))3# Shih, /))%& $/& Aut about 4-,--- ce""s 'er *e"" o( a 241*e"" '"ate in - . m" o( the a''ro'riate com'"ete gro*th me!ium $a!! /-E serum i( it nee!s& $2& Incubate the ce""s at 3% o9 in a 9D2 incubator unti" the ce""s are .-18-E con("uent $about 2- hours, !e'en!ing on the ce""s& $3& 4i"ute 3 g 4,A into 2. " me!ium *ithout serum (or each *e"" an! mi5 $4& 4i"ute 3 " Li'o(ectamine Ceagent into 2. " me!ium *ithout serum (or each *e"" an! mi5 $.& 9ombine !i"ute! 4,A $Ste' 3& an! Li'o(ect1 amine Ceagent $Ste' 4& an! incubate at room tem'erature (or 3- min In this ste' the 4,A1"i'osome com'"e5es are (orme! $?& Ce'"ace the me!ium in the ce""s *ith - 2 m" trans(ection me!ium *ithout serum $%& A!! - /. m" me!ium *ithout serum to the tube containing the com'"e5es (or each *e"" $8& Incubate the ce""s *ith the com'"e5es (or about /- hours at 3%o9 in a 9D2 incubator 8he incubating time *i"" be ("e5ib"e by the ce"" ty'e $)& A!! - 4 m" gro*th me!ium containing !oub"e the 2F norma" concentration o( the serum *ithout removing the trans(ection mi5ture $/-& Ce'"ace the me!ium *ith (resh, com'"ete me!ium at 2- hours (o""o*ing the start o( trans(ection i( continue! ce"" gro*th is re:uire! $//& Assay ce"" e5tracts (or transient gene e5'ression at 241%2 hours a(ter trans(ection, !e'en!ing on the ce"" ty'e an! 'romoter activity $/2& 8o obtain stab"e trans(ectants, 'assage the ce""s /0/- into the se"ective me!ium a(ter %2 hours o( trans(ection (or the re'orter gene trans(ecte! 3.2 Calcium-phosphate mediated 8o get a better !escri'tion, the (o""o*ing 'rotoco" is using the human inter"eu7in12 gene trans(er into cu"ture! rat myocytes as the e5am'"e manua" 3.2.1 Rat heart uscle cells are !ri arily culture" ed: $/& A!u"t rats are sacri(ice! by !eca'itation *ith a !eca'itator $2& Cat hearts are move! out an! "e(t atria are iso"ate! un!er steri"e con!ition $3& 8issue is trans(erre! to a (resh steri"e 'hos'hate bu((ere! so"ution $A<S& an! rinse $4& 8rans(er to a secon! !ish an! !issect o(( un*ante! tissue such as (at or necrotic materia" an! cho' (ine"y *ith crosse! sca"'e"s to about / mm cubes $.& 8rans(er by 'i'ette $/- G 2- m" *ith *i!e ti'& to a /.1m" steri"e centri(uge tube $?& Hash by resus'en!ing the 'ieces in A<S, trans(er the cho''e! 'ieces to the try'sini;ation ("as7, an! a!! / m" try'sin so"ution $- 2.E& 'er /-- mg tissue Incubate the tissue in try'sin so"ution (or /2 hours at 4o9 then *ash *ith A<S (or 3 times

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Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique $%& A!! / m" try'sin so"ution $- 2.E& 'er /-- mg tissue, *ith / mg=m" e"astase an! / mg=m" co""agenase then stir at about 2-- r'm (or 3- min at 3? .o9 $8& A""o*ing the 'ieces to sett"e, co""ect su'ernatant, centri(uge at a''ro5imate"y .-- g (or . min, resus'en!ing 'e""et in /- m" me!ium *ith /-E serum $B<S& $3ibco <CL Li(e 8echno"ogies, Inc , 3ran! Is"an!, ,I, USA&, an! store ce""s on ice $)& A!! (resh try'sin to 'ieces an! continue to stir an! incubate (or a (urther 3- min Ce'eat ste's ? G 8 unti" com'"ete !isaggregation occurs or unti" no (urther !isaggregation is a''arent $/-& 9o""ect an! 'oo" chi""e! ce"" sus'ensions, an! count by hemocytometer $//& 4i"ute to /-? 'er m" in gro*th me!ium an! see! as many ("as7s as are re:uire! *ith a''ro5imate"y 2 5 /-. ce""s 'er m" or set u' a range o( concentrations (rom about /- mg tissue 'er m" $/2& Aut into 9D2 incubator *ith 3? .o9 $/3& 9u"ture me!ium use! is Me!ium /)) *ith /-E B<S A"" the so"utions use! contain - / mg=m" o( anti1biotic am'ici""in $Sigma, St Louis, MD, USA& 3.2.2 #acteria $ulture $Sambroo7, /)8)# Bre!eric7, /))2&: %1& Growth of E. coli: 4isso"ve ). co i in - 3 m" L< '"us tetracyc"ine $2 mg=m"& me!ium, trans(er it into a tube containing . m" L< '"us tetracyc"ine $2 mg=m"& me!ium, 3%o9 overnight, then (ree;e it at 1%-o9 %2& 'ar(estin) *. coli: A Strea7 an inocu"um across one si!e o( a '"ate Cesteri"i;e an inocu"ating "oo' an! strea7 a sam'"e (rom the (irst strea7 across a (resh 'art o( '"ate, then incubate at 3%o9 unti" co"onies a''ear $overnight& < 8rans(er a sing"e bacteria" co"ony into 2 m" o( L< me!ium containing tetracyc"ine $2 mg=m"& in a "oose"y ca''e! /.1m" tube 3%o9 overnight *ith vigorous sha7ing 9 Aour / . m" o( the cu"ture into a micro(uge tube 9entri(uge at /2,---g (or 3- secon!s at 4o9 in a micro(uge Store remain!er at 4o9 4 Cemove the me!ium by as'iration %3& +ysis of *. coli and !urification of !las id: Cesus'en! ). co i 'e""et in /-- " o( ice1co"! So"ution I $.- mM g"ucose, 2. mM 8ris19", 'H 8 -, /- mM E48A, 'H 8 -& A!! 2-- " o( (resh"y 're'are! So"ution II $- 2 , ,aDH, /E S4S&, inverting the tube ra'i!"y . times 4o not vorte5 Store at 4o9 A!! /.- " ice1co"! So"ution III $. M 'otassium acetate ?- m", g"acia" acetic aci! // . m", H2D 28 . m"&, on ice (or 31. min 9entri(uge at /2,---g (or /- min, at 23o9 Aour su'ernatant into JIA're' co"umn $si"icon ge" co"umn, Jiagen 9om'any, USA& 9entri(uge at /2---g (or / min an! !iscar! ("o* through Hash the co"umn *ith - %. m" AE bu((er $.. m" o( . mM Mo's1KDH, 'H % .1%, - %. mM ,a9" '"us 22- m" o( ethano"& 9entri(uge / min at /2---g an! !iscar! ("o* through A"ace co"umn in / . m" microcentri(uge tube A!! .- " o( the 4EA9 H2D in the center o( the co"umn, stan! (or / min, centri(uge at /2---g (or / min 8a7e / " o( 4,A $'"asmi!&, a!! )) " o( 8E bu((er, 'H 8 -, measure 4,A concentration at D42?- nm an! D428- nm $D42?nm=D428- nm shou"! be M/ %& Ce!isso"ve the 4,A in .- " o( 8E $'H 8 -& containing 4,Aase1(ree 'ancreatic C,Aase $2- g=m"& @orte5 brie("y Store at 12-o9 9a"cu"ate the concentration o( the '"asmi! 4,A0 / D42?- nm N .- g o( '"asmi! 4,A=m" Store the 4,A in a"i:uots at 12-o9

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3.2.3 Transfer hu an interleu,in"2 )ene into rat heart uscle cells: %1& Transferred )ene: Human inter"eu7in12 $IL1 2& gene c"one! in '"asmi! '<C322 inserte! in ). co i can be bought (rom American 8y'e 9u"ture 9o""ection $A899, Coc7vi""e, M4, USA& %2& Transfection: 2/-% o( heart musc"e ce""s sus'en!e! in - 2 m" me!ium are see!e! into a tissue cu"ture chamber 481%2 hours "ater, remove me!ium an! a!! - 2 m" (resh me!ium, then a!! - . g o(

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Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique '"asmi! in - -. m" ca"cium 'hos'hate1HEAES1bu((ere! sa"ine, 'H % -, at 3%o9 3.2.- .etection of interleu,in"2: /2O48 hours a(ter the a!!ition o( '"asmi! an! incubation, the amount o( inter"eu7in12 is measure! *ith the in!irect en;yme1"in7e! immunosorbent assay $ELISA& in me!ium Antibo!y o( anti1inter"eu7in12 $human& can be gotten (rom Sigma $Sigma 9hemica" 9o , St Louis, MD, USA& 3.3 .*A*"de/tran ediated 4EAE1!e5tran $!iethy"aminoethy"oethy"1!e5tran& *as use! to intro!uce 'o"iovirus C,A an! S@4- an! 'o"yomavirus 4,As into ce""s in /)?-s $Aagano, /)?.# Mc9utchan, /)?8# Har!en, /)?8& 8here are three 'oints that 4EAE1!e5tran me!iate! trans(ection !i((ers (rom ca"cium 'hos'hate co'reci'itation $/& It is use! (or transient trans(ection $2& It *or7s more e((icient"y *ith ce"" "ines o( <S91/, 9@1/ an! 9DS, etc $3& It is more sensitive 8he 4EAE1!e5tran me!iate! trans(ection cou"! be !one by the (o""o*ing ste's0 $/& Harvest e5'onentia""y gro*ing ce""s by try'sini;ation an! trans(er then into ?-1mm tissue cu"ture !ishe! at a !ensity o( /-. ce""s=!ish $2& A!! . m" com'"ete gro*th me!ium $3& Incubate 24 hours at 3%oC #ith 53 C42. $4& Are'are 4,A=4EAE1!e5tran=8<S14 so"ution by mi5ing 2 mg o( su'eroi"e! '"asmi! 4,A into / g5& ()*)%de+tran in T/S%(. (5) 2e&o6e &ediu& and #ash tree ti&es #ith 7/S and t#ice #ith T/S%(. $?& A!! 4,A=4EAE1!e5tran=8<S14 so"ution 2." $%& Incubate ?- min at 3%o9 *ith .E 9D2 $8& Cemove 4,A=4EAE1!e5tran=8<S14 so"ution $)& Hash *ith 8<S14 three time an! A<S t*ice $/-& A!! . m" me!ium su''"emente! *ith serum an! ch"oro:uine $- / mM& $//& Incubate 4 hours at 3%o9 *ith .E 9D2. (12) 2e&o6e &ediu&. $/3&8ash #ith seru&%free &ediu& three ti&es. $/4& A!! to ce""s . m" o( me!ium su''"ement *ith serum, an! incu!ate ,9 hours at 3:oC #ith 53 C42. $/.& Harvest the ce""s a(ter the 48 hours trans(er % ction $/?& Ana"y;e C,A or 4,A by hybri!i;ation, or ana"y;e e5'resse! 'rotein by ra!iommunoassay, immunob"otting, immuni'reci'itation, or by en5;y% moatic acti6it0 in ce e+tract. 3.- Electroporation Au"se e"ectrica" (ie"!s can be use! to intro!uce 4,A into ce""s o( anima", '"ant an! bacteria Bactors that in("uence e((iciency o( trans(ection by e"ectro'oration0 a''"ie! e"ectric (ie"! strength, e"ec tric "u se ength, te&"erature, (N* confor&ation, (N* concentration, and ionic co&"osition of transfection &ediu&, etc. Ste!s of the electro!oration transfection: $/& Harvest ce""s in the mi!1 to "ate1"ogarithmic 'hase o( gro*th $2& 9entri(uge at .-- g $2--- r'm& (or . min at 4oC. (3) 2esus"end ce s in gro#th &ediu& at concen% tration o( / P /-: ce s5& . $4& A!! 2- g " as&id (N* in ,0 ce s. $.& E"ectric trans(ect by 3-- @ = /-.- B (or /12 min $?& 8rans(er the e"ectro'orate! ce""s to cu"ture !ish an! cu"ture the ce""s $%& Assay 4,A, C,A or 'rotein an! continuous"y cu"ture the ce""s to get 'ositive ce"" "ines. . 3.0 Polybrene Severa" 'o"ycations, inc"u!ing 'o"ybrene $/,.1 !imethy"1/,.1!ia;aun!ecamethy"ene 'o"y1 methobromi!e& $9haney, /)8?& an! 'o"y1L1ornithine $,ea!, /)).&, have been use! in gene trans(ection *ith the 4MSD enhancement ,orma" ste's are (o""o*ing0 $/& Harvest e5'onentia" ce""s by try'sin;ationin an! re'"ate at a !ensity o( .,--- ce""s=mm2 in /- m" MEM1 containing /-E (eta" ca"( serum $2& Incubate 24 hours at 3%o9 in .E 9D2. (3) 2e" ace &ediu& #ith 3 & "re%#ar&ed &ed% ium containing serum, /- g 4,A an! 3- g 'o"ybrene $3%o9& Mi5 the me!ium be(ore a!!ing 'o"ybrene $4& Incubate /2 hours *ith a gent sha7e each hour $.& Cemove me!ium an! a!! . m" 3-EQ 4MSD in serum1containing me!ium

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Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique $?& A(ter 4 min incubation, as'irate the 4MSD so"ution Hash the ce""s t*ice *ith *arme! $3%o9& serum1(ree me!ium, an! a!! /- m" com'"ete me!ium containing /-E (eta" ca"( serum $%& Incubate 48 hours at 3%o9 in .E 9D2. (9) )+a&ine the ce s e6er0da0 after the transfer% ction $)& Bor stab"e trans(ection, continue incubate 3 *ee7s *ith changing me!ium every 2 !ays 3.1 2irus @iruses are high"y a!a'te! to the 'rocess o( gene trans(er @ira" vectors have the abi"ity to trans(er 4,A to a high (raction o( ce""s, but using virus as the vector *i"" be 'otentia""y arouse cancer "eu7aemia $9ava;;ana19a"vo, 2--4& 9ommon vectors use! (or gene trans(er in ce"" cu"ture are !erive! (rom retroviruses A!enovirus an! other agents are use! (or the gene !e"ivery 3.3 Biolistics (Gene )un4 or called icro!article bo bard ent& Some ce""s, tissues an! intrace""u"ar organe""es are im'ermeab"e to (oreign 4,A, es'ecia""y '"ant ce""s bio"istics, inc"u!ing 'artic"e bombar!ment, is a common"y use! metho! (or genetic trans(ormation o( '"ants an! other organisms 8o reso"ve this 'rob"em in gene trans(er, the gene gun *as ma!e by K"ein at 9orne"" University in /)8% $K"ein, /)8%# Ki77ert, 2--.& Dn the gene gun techni:ue, K"ein an! San(or! et a" 'ub"ishe! 'a'ers, obtaine! 'atents an! (orme! a com'any ca""e! <io"istics $K"ein, /)8%& 8he gene gun is 'art o( the gene trans(er metho! ca""e! the bio"istic $a"so 7no*n as bioba"istic or 'artic"e bombar!ment& metho! In this metho!, 4,A or C,A a!here to bio"ogica" inert 'artic"es $such as go"! or tungsten& <y this metho!, 4,A1'artic"e com'"e5 is 'ut on the to' "ocation o( target tissue in a vacuum con!ition an! acce"erate! by 'o*er(u" shot to the tissue, then 4,A *i"" be e((ective"y intro!uce into the target ce""s Uncoate! meta" 'artic"es cou"! a"so be shot through a so"ution containing 4,A surroun!ing the ce"" thus 'ic7ing u' the genetic materia" an! 'rocee!ing into the "iving ce""s 8he e((iciency o( the gene gun trans(er cou"! be !e'en!e! on the (o""o*ing (actors0 ce"" ty'e, ce"" gro*th con!ition, cu"ture me!ium, gene gun ammunition ty'e, gene gun settings an! the e5'erimenta" e5'eriences, etc <rie("y (or gene gun 'ractice, the target ce""s or tissues on the 'o"ycarbonate membranes cou"! be 'ositione! in a <io"istic A4S1/---=HE Aartic"e 4e"ivery System $<io1Ca! Laboratories 3mbH, MRnchen, 3ermany& <io"istic 'arameters are /. in Hg o( chamber vacuum, target !istance o( 3 cm $stage /&, )-- 'si to /8-- 'si 'artic"e acce"eration 'ressure, an! / - m !iameter go"! microcarriers $<io1Ca!, USA& 3o"! microcarriers are 're'are!, an! circu"ar '"asmi! 4,A is 'reci'itate! onto the go"! using metho!s recommen!e! by <io1Ca! *ith the (o""o*ing0 - ? mg o( go"! 'artic"es carrying O. g o( '"asmi! 4,A is use! 'er bombar!ment 8he !etai" 'rotoco" (or the gene gun trans(ection is !escribe! as (o""o*s0 $/& Are'are go"! or tungsten 'artic"es0 ?- mg go"! or tungsten in / m" %-E ethano", centri(uge at /-,--r'm (or /- secon!s an! co""ect 'artic"es, an! *ash *ith H2D three times by centri(ugation $2& Are'are 4,A1coate! 'artic"es0 Mi5 .- " $about 3 mg& meta", 2 . " '"asmi! 4,A $about 2 . g&, 9a9"2 .- " $2 . M&, s'ermi!ine 2- " $- /M& @orte5 an! stan! (or . min 9entri(uge, remove su'ernatant, an! a!! /4- " %-E ethano" over the 'e""ete! 'artic"es, an! re'eat the ethano" an! centri(ugation three times, then a!! .- " ethano" $3& A"ace a macrocarrier in the meta" ho"!er o( gene gun an! *ash t*ice *ith ethano" $4& @orte5 an! s'rea! - . mg 'e""et s"urry on the macrocarrier $.& Loa! the macrocarrier into the gene gun, an! shoot it Ce'eat the shoot unti" a"" the areas are shot $?& Bor transient e5'ression, e5amine ce""s 48 hours a(ter the shooting, by immuno"ogy or other metho!s $%& Bor stab"e trans(ection, continue cu"ture the trans(ecte! ce""s or tissues In our stu!ies, *e !i! gene trans(ection *ith the se"(1!esign 9D2 'ro'e""e! gene gun $2-- 'si, !istance 3 cm *ith 4-- mesh ny"on screen& using tungsten 'artic"e $?-- nm !iameter& coate! *ith '"asmi! e5'ressing anti1am'ici""in gene, the '"asmi! *ith anti1am'ici""in gene *as trans(erre! into E 9o"i ce""s

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Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique 3.5 +aser transfection As the e5am'"e o( our e5'eriments, U@ e5cimer "aser $Pe9"2, 3-8 nm& is use! in the gene trans(ection $. min by a - % - ), / 4 or 2 - mm !iameter (iber *ith ("uence o( 4. an! ?- mS=mm2 1 rea" "aser energy 2 3, . ), /3 /, 32 - mS='u"se, 2. H;& $9@P13-- E5cimer Laser System, S'ectranetics 9or'oration, 9o"ora!o S'rings, 9D, USA& A"so, *e use! to ma7e e5'eriments *ith ,!0Iag, Ho0Iag in the gene trans(ection A"" the metho!s o( e5cimer, ,!0Iag an! Ho0Iag "aser trans(ection are e((ective 3.6 Transfection enhanced by ele(ated te !era" ture Dur stu!ies sho* that high tem'erature enhances the gene trans(ection In our e5'eriments, rat heart musc"e ce""s *ere cu"ture! in me!ium /)) *ith /-E B<S an! human aorta smooth musc"e ce""s *ere cu"ture! in B/2K me!ium Human inter"eu7in12 gene *as trans(ecte! into rat heart ce""s an! s*ine gro*th hormone gene *as trans(ecte! into human aorta smooth musc"e ce""s by ca"cium 'hos'hate co'reci'itation at various tem'eratures0 23T9, 3%T9 an! 43T9 8rans(ecte! inter"eu7in12 an! s*ine gro*th hormone e5'ressions *ere !etecte! using an in!irect ELISA 8he heate! cu"ture! rat myocytes ha! a signi(icant"y higher e5'ression o( the trans(ecte! inter"eu7in12 gene Ambient tem'erature rise to 43 o9 (or u' to 3- min 'rovi!e! greater transient trans(ection o( the inter"eu7in12 gene *hen com'are! to ambient tem'eratures at 3%o9 an! 23o9 $'U- -/& 8he greatest e((ects occurre! *ithin /- min o( incubation an! 'ersiste! u' to 3- min 8hese resu"ts suggest that even a (e* !egrees o( ambient tem'erature rise can signi(icant"y increase gene trans(er into musc"e ce""s 8his may be o( va"ue *hen using gene thera'y *ith trans(ection 'roce!ures $Ma, 2--4b# Ma, 2--4c& 3.17 8lant )ene transfer Agricu"ture an! '"ant bree!ing re"ie! so"e"y on the accumu"ate! e5'erience o( generations o( (armers an! bree!ers that is, on se5ua" trans(er o( genes bet*een '"ant s'ecies Ho*ever, !eve"o'ments o( '"ant mo"ecu"ar bio"ogy an! genomics no* give us access to 7no*"e!ge an! un!erstan!ing o( '"ant genomes an! the 'ossibi"ity o( mo!i(ying them 8here are t*o most 'o*er(u" techno"ogies (or trans(erring gene into '"ants0 Agrobacterium1me!iate! trans(ormation an! bio"istics As '"ants have ce"" *a"", the bio"istics is very use(u" in the '"ant gene trans(er $Casco13aunt, 2--/& - .iscussion 8he current century *i"" bring tremen!ous changes to the science, techno"ogy, an! the 'ractice o( me!icine $Lushai, 2--2& 3ene thera'y is 'art o( a gro*ing (ie"! in mo"ecu"ar me!icine, *hich *i"" gain im'ortance in the treatment o( human !iseases $3unther, 2--.& As a critica" to'ic, gene trans(ection gives 'eo'"e the ho'e to treat many !iseases but it a"so cou"! create !angerous s'ecies in the earth, so that it attracts '"enty attention by the *ho"e human society $Lan;a, 2--2& 8his sim'"y means that the success o( gene trans(er techni:ue *i"" be bene(it (or the civi"i;ation, an! a"so create the !anger (or the "i(e in the earth either $Schiemann, 2--3& 3ene trans(ection 'roce!ures are use! in the critic 'roce!ure anima" c"one $9hesne, 2--2# Heyman, 2--2&, an! the anima" c"one is cha""enge! by the re"igious grou's an! ethnic e5tremists $Hou!ebine, 2--3& As our 'ersona" vie*s, no matter ho* big cha""enges (rom *hatever as'ects, the gene trans(ection an! anima" c"one *i"" !eve"o' :uic7"y 8he *or"! is a com'"e5 '"ace com'ose! by !i((erent 'eo'"e Bor the science an! techno"ogy such as gene trans(er an! anima" c"one, no country can 'revent other countries (rom the 'ursuing He nee! to !eve"o' the techni:ue even i( the techni:ue cou"! be use! in the !anger action, an! *e nee! to consi!er the socia" e((ects o( a techni:ue *hen *e !eve"o' it either Science !eve"o'ment *i"" be bene(it to a"" the human society As the gene thera'y !eve"o'ing, many more !es'erate !iseases cou"! be cure! an! many human "ivings cou"! be save!, such as the "i(e o( Ao'e Lohn Aau" II an! 8erri Schin!"er1Schiavo Ho'e that the gene trans(er techni:ues !escribe! in this artic"e cou"! be use(u" (or the researches in the gene thera'y (ie"! an! he"' to a!vance the "i(e science stu!y $orres!ondence to: Hongbao Ma <4/- 9"inica" 9enter Michigan State University

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Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique East Lansing, MI 48824, USA 8e"e'hone0 $./%& 4321-?23 Emai"0 hongbao@msu e!u References
+/2 Arat S, C;uci!"o SL, 3ibbons L, Miyoshi K, Stice SL Aro!uction o( transgenic bovine embryos by trans(er o( trans(ecte! granu"osa ce""s into enuc"eate! oocytes Mo" Ce'ro! 4ev 2--/#?-$/&02-1? Avery D8, MacLeo! 9M, Mc9arty M Stu!ies on the chemica" nature o( the substance in!ucing trans(ormation o( 'neumococca" ty'es L E5' Me! /)44#%)0/3%1.8 <or!ignon @, Keyston C, La;aris A, <i"o!eau AS, Aontes LH, Arno"! 4, Becteau 3, Kee(er 9, Smith L9 8ransgene e5'ression o( green ("uorescent 'rotein an! germ "ine transmission in c"one! ca"ves !erive! (rom in vitro1trans(ecte! somatic ce""s <io" Ce'ro! 2--3#?8$?&02-/3123 9am'be"" KH ,uc"ear trans(er in (arm anima" s'ecies Semin 9e"" 4ev <io" /)))#/-$3&024.1.2 9ava;;ana19a"vo M, 8hrasher A, Mavi"io B 8he (uture o( gene thera'y ,ature 2--4#42%$?)%%&0%%)18/ 9haney H3, Ho*ar! 4C, Ao""ar! LH, Sa""ustio S, Stan"ey A High1(re:uency trans(ection o( 9HD ce""s using 'o"ybrene Somat 9e"" Mo" 3enet /)8?#/2$3&023%144 9hesne A, A!enot A3, @ig"ietta 9, <aratte M, <ou"anger L, Cenar! LA 9"one! rabbits 'ro!uce! by nuc"ear trans(er (rom a!u"t somatic ce""s ,at <iotechno" 2--2#2-$4&03??1) 3ri((ith B 8he signi(icant o( 'neumocco'a" ty'es Hyg L /)28#2%0//3 3unther M, Hagner E, Dgris M S'eci(ic targets in tumor tissue (or the !e"ivery o( thera'eutic genes 9urr Me! 9hem Anti 9anc Agents 2--.#.$2&0/.%1%/ Ha*"ey1,e"son A, 9iccarone @, 3ebeyehu 3, Lessee L, Be"gner A BD9US /))3#/.0%3 Hershey A4, 9hase 4 In!e'en!ent (unctions o( vira" 'rotein an! nuc"ei aci! in gro*th o( bacterio'hage L 3en Ahysio" /).2#3?03)1.? Heyman I, Vhou J, Lebourhis 4, 9havatte1Aa"mer A, Cenar! LA, @ignon P ,ove" a''roaches an! hur!"es to somatic c"oning in catt"e 9"oning Stem 9e""s 2--2#4$/&04%1.. Hou!ebine LM Anima" transgenesis an! 9"oning Lohn Hi"ey W Sons, Inc Hobo7en, ,L, USA, 2--30341%3 Lan;a CA, 4resser <L, 4amiani A 9"oning ,oahXs ar7, in Un!erstan!ing 9"oning Scienti(ic American, Inc an! <yron Aress @isua" Aub"ications, Inc, 2--202413. +/.2 Ki77ert LC, @i!a" LC, Ceisch <I Stab"e trans(ormation o( '"ant ce""s by 'artic"e bombar!ment=bio"istics Metho!s Mo" <io" 2--.#28?0?/1%8 +/?2 K"ein 8M, Ho"( E4, Hu C, San(or! L9 Hugh1ve"ocity micro'roSeecti"es (or !e"ivering nuc"eic aci!s into "iving ce""s ,ature /)8%X32%0%-13 +/%2 Lorence A, @er'oorte C 3ene trans(er an! e5'ression in '"ants Metho!s Mo" <io" 2--4#2?%032)1.+/82 Ma H 8echni:ue o( Anima" 9"one ,ature an! Science 2--4$a&#2$/&02)13. +/)2 Ma H, 9hi 9, Abe"a 3S Increase in ambient tem'erature enhances gene trans(er into human smooth musc"e ce""s BASE< Lourna" 2--4$b&#/8$8&092)3 +2-2 Ma H, 9hi 9, Abe"a 3S Increase! ambient tem'erature enhances human inter"eu7in12 gene trans(er into cu"ture! myocytes Lourna" o( Investigative Me!icine 2--4$c&#.2$2&0S3)+2/2 Mc9utchan LH, Aagano LS Enchancement o( the in(ectivity o( simian virus 4- !eo5yribonuc"eic aci! *ith !iethy"aminoethy"1 !e5tran L ,at" 9ancer Inst /)?8#4/$2&03./1% +222 ,ea! MA, Mc9ance 4L Ao"y1L1ornithine1me!iate! trans(ection o( human 7eratinocytes L Invest 4ermato" /)).#/-.$.&0??81%/ +232 Aagano LS, @aheri A Enhancement o( in(ectivity o( 'o"iovirus C,A *ith !iethy"aminoethy"1!e5tran $4EAE14& Arch 3esamte @irus(orsch /)?.#/%$3&04.?1?4 +242 Casco13aunt S, Ci"ey A, 9anne"" M, <arce"o A, La;;eri AA 2--/ Aroce!ures a""o*ing the trans(ormation o( a range o( Euro'ean e"ite *heat $Triticu& aesti6u& L & varieties 6ia 'artic"e bombar!ment L E5' <ot 2--/#.2$3.%&08?.1%4 +2.2 Sambroo7 L, Cusse"" 4H Mo"ecu"ar c"oning0 a "aboratory manua" 9o"! S'ring Harbor Laboratory Aress, 9o"! S'ring Harbor, ,I, USA 2--/#3$/?&0/? /1/? ?2 +2?2 Schiemann L ,e* science (or enhance! biosa(ety Environ <iosa(ety Ces 2--3#2$/&03%14/ +2%2 Shih AL, Evans K, Schi((er"i KA, 9iccarone @, Lichaa B, Masou! ML, Ha*"ey1,e"son A BD9US /))%#/)0.2 +282 Umemoto I, Sasa7i S, KoSima I, Kubota H, Kane7o 8, Hayashi I, Kohri K 3ene trans(er to mouse testes by e"ectro'oration an! its in("uence on s'ermatogenesis L An!ro" 2--.#2?$2&02?41%/ +2)2 U;ogara S3 8he im'act o( genetic mo!i(ication o( human (oo!s in the 2/st century0 a revie* <iotechno" A!v 2---#/8$3&0/%)12-? +3-2 Har!en 4, 8horne H@ 8he in(ectivity o( 'o"yoma virus 4,A (or mouse embryo ce""s in the 'resence o( !iethy"aminoethy"1 !e5tran L 3en @iro" /)?8#3$3&03%/1%

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