US 20010044150A1

( 19 ) United Sta tes

( 12) Pa tent Applica tion Publica tion ( 10) Pub. N o. : US 2001/ 0044150 A1
Hohn et a l. ( 43 ) Pub. D a te: N OV. 22, 2001
( 54) METHOD FOR TRAN SFORMATION OF Rela ted US. Applica tion D a ta
AN IMAL CELLS
( 6 3 ) Continua tion of a pplica tion N o. PCT/ EP9 9 / 05256 ,
? led on J ul. 22, 19 9 9 .
( 7 6 ) Inv entor s : B a r ba r a Hohn, Ar les heim ( CH) ; Luca ( 3 0) For eig n Applica tion Pr ior ity D a ta
Ros s i, N eucha tel ( CH) ; Alicj a
Ziem ienowicz, Gda ns k ( PL) ; B is er ka J ul. 24, 19 9 8 ( GB ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 816 13 83
Relic, Lieg e B E
( ) Publica tion Cla s s i? ca tion
Cor r es pondence Addr es s :
51 Int. Cl. 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . C12N 15/ 86 ; C12N 15/ 87
g g y lg g g lg g lg g m m E523 US. Cl. . . . . . . . . . . . . . . . . . . . . . . . . . - 43 5/ 459 . 43 5/ 456 . 43 5/ 46 1
PATEN T AN D TRAD EMARK D EPT ( 57 ) AB STRACT
56 4 MORRIS AVEN UE The pr es ent inv ention r ela tes to a m ethod f or intr oducing
SUMMIT, N J 07 9 011027 nucleic a cids into cells f or eg pr oducing tr a ns iently tr a ns
f ected or s ta bly tr a ns f or m ed a nim a l cells by us ing a s pe
ci? ca lly des ig ned nucleic a cid/ pr otein com plex com pr is ing
( 21) Appl. N o. : 09 / 7 6 8, 412 in oper a ble linka g e to a n ex pr es s ible D N A or to a n olig o
nucleotide a Vir D 2 pr otein, pr ef er a bly tog ether W ith a Vir E2
( 22) Filed: J a n. 24, 2001 pr otein.
US 2001/ 0044150 A1
METHOD FOR TRAN SFORMATION OF AN IMAL
CELLS
FIELD OF THE IN VEN TION
[ 0001] The pr es ent inv ention g ener a lly r ela tes to the tr a ns
f or m a tion of euka r y otic cells , pa r ticula r ly a nim a l cells , W ith
ex og enous nucleic a cids a nd to the g ener a tion of tr a ns g enic
or g a nis m s g ener a ted f r om s uch cells . Mor e pa r ticula r , the
pr es ent inv ention r ela tes to a m ethod f or intr oducing nucleic
a cids into cells f or pr oducing tr a ns iently tr a ns f ected/ tr a ns
f or m ed or s ta bly tr a ns f or m ed cells by us ing a s peci? ca lly
des ig ned nucleic a cid/ pr otein com plex , a s W ell a s to cells
tr a ns f ected or tr a ns f or m ed ther eby .
B ACKGROUN D OF THE IN VEN TION
[ 0002] Sev er a l m ethods ha v e been dev eloped f or intr o
ducing ex og enous D N A m olecules into euka r y otic cells in
or der to ta ke a dv a nta g e of the W ides pr ea d bene? ts a r is ing
f r om the a pplica tion of r ecom bina nt D N A technolog y to the
pr oduction of tr a ns iently tr a ns tected/ tr a ns f or m ed cells a s
W ell a s to tr a ns g enic cells a nd or g a nis m s g ener a ted f r om
s uch cells . Thes e m ethods include phy s ica l, non- biolog ica l
s y s tem s s uch a s electr opor a tion, m icr oinj ection, ca lcium
phos pha te or poly ethy lene g ly col ( PEG) m edia ted D N A
upta ke or cell f us ion, a nd m icr opr oj ectile bom ba r dm ent, a nd
m odi? ed biolog ica l s y s tem s s uch a s Ag r oba cter ium - m edi
a ted T- D N A tr a ns f er to pla nt cells ( f or a g ener a l ov er v ieW ,
s ee cha pter s 2 a nd 3 of Pla nt Genetic Tr a ns f or m a tion a nd
Gene Ex pr es s ion, A La bor a tor y Ma nua l , ed. by D r a per , J .
et a l. , pub. by B la ckwell Scienti? c Publica tions ( 19 88) ; s ee
a ls o Potr y kus , et a l. , D ir ect Gene Tr a ns f er : Sta te of the Ar t
a nd Futur e Potentia l , Pla nt Mol. B iol. Rep. 3 : 117 - 128
( 19 85) ) .
[ 0003 ] The m ethods W hich ha v e been dev eloped ha v e
a lloW ed the s ta ble tr a ns f or m a tion of a W ide v a r iety of cells
a nd or g a nis m s W ith ex og enous D N A. In pa r ticula r , the
dev elopm ent of phy s ica l techniq ues s uch a s biolis tics ha s
ov er com e a ppa r ent hos t- r a ng e lim ita tions im pos ed by bio
log ica l s y s tem s . HoW ev er , a com m on de? ciency of thes e
phy s ica l m ethods is tha t they do not pr ov ide a ny m ea ns f or
or der ed integ r a tion of the deliv er ed nucleic a cid into the cell
g enom e. Cons eq uently thes e m ethods m us t depend upon
uncontr olled integ r a tion of the deliv er ed nucleic a cid by
poor ly under s tood m echa nis m s , ca us ing ex og enous D N A to
be integ r a ted a s m ultiple copies of r a ndom f r a g m ents us u
a lly a t a s ing le s ite in the cell g enom e.
[ 0004] Im pr ov ing the pr edicta bility of s ta ble tr a ns f or m a
tion ev ents a r is ing f r om the phy s ica l intr oduction of ex og
enous nucleic a cid into the cell W ould s ig ni? ca ntly im pr ov e
the utility a nd ov er a ll ef f iciency of thes e pr oces s es f or
pr oducing g enetica lly s ta ble tr a ns f or m ed cells or or g a nis m s
ex hibiting s ta ble ex pr es s ion of tr a ns g enes . One a ppr oa ch
W hich ha s been ta ken to a ccom plis h this g oa l ha s been to
com bine pr oteins W hich pr om ote tr a ns f or m a tion a nd/ or inte
g r a tion in biolog ica l s y s tem s W ith non- biolog ica l deliv er y
techniq ues . In or der to a chiev e the des ir ed ef f ect, it ha s been
cons ider ed neces s a r y to a s s ocia te the pr oteins them s elv es
W ith the ex og enous D N A m olecules pr ior to deliv er y to the
tr a ns f or m a tion ta r g et cell, thus m im icking a s clos ely a s
pos s ible the biolog ica l s y s tem f r om W hich the pr oteins a r e
der iv ed ( s ee, eg inter na tiona l a pplica tion no. PCT/ EP9 4/
0256 6 to Hohn et a l. publis hed Feb. 23 , 19 9 5 a s W O
N ov . 22, 2001
9 5/ 0547 1; inter na tiona l a pplica tion no. PCT/ US9 5/ 07 543 to
Cona r y , J . et a l. publis hed D ec. 21, 19 9 5 a s W O 9 5/ 3 46 47 ) .
[ 0005] The Ag r oba cter ium pla nt tr a ns f or m a tion s y s tem
m entioned a bov e is W idely us ed f or the s ta ble tr a ns f or m a
tion of hig her pla nts . In this s y s tem g enes to be tr a ns f er r ed
a r e ca r r ied by the T- D N A, a W ell- de? ned r eg ion of the
Ag r oba cter ium Ti pla s m id. The Ti pla s m id a ls o conta ins a
v ir ulence ( v ir ) r eg ion, W hich encodes pr oteins inv olv ed in
the tr a ns f or m a tion v ia Ag r oba cter ium of pla nt cells . At lea s t
one of thes e pr oteins , Vir D 2 is inv olv ed in ta r g eting to the
pla nt nucleus a nd integ r a tion into the pla nt g enom e ( Tinla nd
et a l. ( 19 9 2) Pr oc. N a tl. Aca d. Sci. USA 89 : 7 442; Tinla nd
et a l. ( 19 9 5) EMB O J . 14: 3 588- 3 59 5) . W O 9 5/ 0547 1, the
contents of W hich is her eW ith incor por a ted by r ef er ence,
dis clos es a m ethod f or pr oducing s ta bly tr a ns f or m ed pla nt
m a ter ia l, including phenoty pica lly nor m a l looking a nd pr ef
er a bly f er tile pla nts , W hich m ethod does not inv olv e Ag r o
ba cter ium tr a ns f or m a tion. In pa r ticula r , it dis clos es a s pe
ci? ca lly a da pted nucleic a cid/ pr otein com plex com pr is ing a
chim er ic r ecom bina nt nucleic a cid, W hich m a y com pr is e, f or
ex a m ple, a n ex pr es s ible D N A or a n olig onucleotide oper
a bly linked to s uita ble pla nt ex pr es s ion s ig na ls inv olv ing
pr om oter a nd ter m ina tion s eq uences a nd cov a lently a s s oci
a ted ther eW ith a Vir D 2, a nd, optiona lly , Vir E2 pr otein units .
HoW ev er , the tea ching does not m ention potentia l a pplica
bility of this tr a ns f or m a tion techniq ue to the ? eld of a nim a l
cells . Fur ther m or e, it does not concer n us ing s peci? ca lly
des ig ned olig onucleotides a s nucleic a cid com ponent of s a id
com plex in a n a ntis ens e- , a ntig ene- or olig oZy m e- a ppr oa ch
f or ( tr a ns ient) tr a ns f ection/ tr a ns f or m a tion of euka r y otic
cells .
[ 0006 ] Since ther e ex is ts a continuous need f or f ur ther
techniq ues W hich a r e us ef ul f or the intr oduction of nucleic
a cids into a nim a l a nd pla nt cells , e. g . olig onucleotides f or
a ntis ens e- or a ntig ene- a ppr oa ches , or f or the per m a nent
tr a ns f or m a tion of a nim a l cells , the obj ect of the pr es ent
inv ention is ther ef or e to pr ov ide a neW m ethod f or intr o
ducing nucleic a cids into euka r y otic cells .
SUMMARY OF THE IN VEN TION
[ 0007 ] The pr es ent inv ention pr ov ides a n im pr ov ed
m ethod f or deliv er ing nucleic a cids a s nucleic a cid/ pr otein
com plex to euka r y otic cells , e. g . olig onucleotides or ex og
enous D N A f or s ta bly tr a ns f or m ing or tr a ns iently tr a ns f ect
ing / tr a ns f or m ing a nim a l, pr ef er a bly m a m m a lia n cells . This
im pr ov ed m ethod f or ex a m ple g ener a lly com pr is es pr ov id
ing to the cell ta r g eted f or tr a ns f ection/ tr a ns f or m a tion a
s peci? ca lly des ig ned nucleic a cid/ pr otein com plex com pr is
ing nucleic a cids s uch a s eg ex og enous D N A or olig o
nucleotide des ir ed to be intr oduced a nd, if des ir ed, to be
integ r a ted in the la ter tr a ns f or m a nt.
[ 0008] For ex a m ple, the pr es ent inv ention pa r ticula r ly
pr ov ides a n im pr ov ed m ethod f or tr a ns iently tr a ns f ecting /
tr a ns f or m ing or f or s ta bly tr a ns f or m ing a nim a l cells W ith
ex og enous nucleic a cids s uch a s eg D N A, W hich com bines
pos itiv e a ttr ibutes of Ag r oba cter ium tum ef a ciens m edia ted
T- D N A tr a ns f er s uch a s hig h- ef ? cient nuclea r ta r g eting a nd
integ r a tion, W ith non- biolog ica l deliv er y m ethods . This
a s pect of the inv ention e. g . com pr is es pr ov iding a n a nim a l
cell W ith the ex og enous D N A f r a g m ent des ir ed to be intr o
duced into the nucleus a nd integ r a ted into the a nim a l cell
g enom e, bounded by T- D N A bor der s or f unctiona l pa r ts
US 2001/ 0044150 A1
ther eof , a long W ith a t lea s t one Ag r oba cter ium - der iv ed pr o
tein tha t ta r g ets s a id f r a g m ent to the nucleus a nd pr om otes
the integ r a tion of the ex og enous D N A into the hos t cell
g enom e. The Ag r oba cter ium - der iv ed pr otein us ed a ccor ding
to the inv ention is s elected f r om the g r oup cons is ting of
Vir D l, Vir D 2, Vir E2, a nd Vir C. Pr ef er a bly , a com bina tion of
Vir D 2 a nd either Vir D l, Vir C, Vir E2, or a s ubcom bina tion
ther eof , is us ed. Mos t pr ef er a bly , us e is m a de of the Ag r o
ba cter ium - der iv ed pr oteins Vir D 2 a nd Vir E2 in com bina
tion, a lthoug h in cer ta in ca s es s ole us e of Vir D 2 m a y be
s uf f icient.
[ 0009 ] Accor ding to the inv ention, the nucleic a cid/ pr otein
com plex com pr is ing the ex og enous nucleic a cid, s uch a s eg
a D N A f r a g m ent bounded by T- D N A bor der s eq uences or
f unctiona l pa r ts ther eof , m a y be deliv er ed to the a nim a l cell
by non- biolog ica l m ea ns s uch a s , but not r es tr icted to,
electr opor a tion, m icr oinj ection, induced upta ke, m icr o
pr oj ectile bom ba r dm ent, or other m ea ns a s a r e knoW n in the
a r t.
[ 0010] In a nother a s pect of the inv ention, a nim a l cells or
tis s ues s ta bly tr a ns f or m ed W ith a dis cr ete D N A f r a g m ent a r e
r eg ener a ted to pr oduce tr a ns g enic a nim a l or g a ns or W hole
a nim a ls tha t s ta bly ex pr es s a des ir ed hom olog ous or heter
olog ous nucleic a cid a nd, in the la tter ca s e, pa s s it on to
pr og eny in W hich s ta ble ex pr es s ion of the tr a ns g ene is
inher ited a s a Mendelia n tr a it.
[ 0011] Fur ther m or e, the pr es ent inv ention pr ov ides nov el
m ea ns f or the in v iv o a nd ex v iv o/ in v itr o tr a ns f or m a tion a nd
integ r a tion or tr a ns ient tr a ns f ection/ tr a ns f or m a tion of ex og
enous nucleic a cids des ir ed to be ex pr es s ed W ithin a nim a l
hos ts or hos t cells , pa r ticula r ly f or the pur pos e of g ene
ther a py .
[ 0012] In a nother a s pect, the pr es ent inv ention s upplies
the pr ocedur es f or intr oducing s m a ll nucleic a cid f r a g m ents
into- a nim a l cells - a nd- pla nt m a ter ia l, f or us e in a ntis ens e,
a ntig ene, olig oZy m e or m uta g enes is technolog y .
D ETAILED D ESCRIPTION OF THE
IN VEN TION
[ 0013 ] The pr es ent inv ention pr ov ides a n im pr ov ed
m ethod f or intr oducing nucleic a cids a s nucleic a cid/ pr otein
com plex to euka r y otic cells , e. g . olig onucleotides or ex og
enous D N A f or s ta bly tr a ns f or m ing a nim a l cells . N ucleic
a cid( s ) a s a com ponent of nucleic a cid/ pr otein com plex
a ccor ding to the pr es ent inv ention m a y be a ny ty pe of s ing le
or double- s tr a nded nucleic a cid, f or ex a m ple RN A, m odi? ed
RN A or D N A, W her ein D N A is the pr ef er r ed f or m . This
im pr ov ed m ethod f or ex a m ple g ener a lly com pr is es pr ov id
ing to the a nim a l cell ta r g eted f or tr a ns f or m a tion a s peci?
ca lly des ig ned nucleic a cid/ pr otein com plex com pr is ing
ex og enous nucleic a cid des ir ed to be integ r a ted a nd
ex pr es s ed in the la ter tr a ns f or m a nt. In this contex t, the ter m s
ex pr es s ed or ex pr es s ible us ed thr oug hout the s peci? ca
tion s ha ll m ea n tha t a g iv en nucleic a cid ca n a t lea s t s er v e
a s ta r g et f or tr a ns cr iption W ithin the nucleus of a cell to be
tr a ns iently or per m a nently tr a ns f ected or tr a ns f or m ed. The
ter m T- D N A bor der ( s ) or f unctiona l pa r t( s ) ther eof s ha ll
encom pa s s the W hole T- D N A bor der s eq uence( s ) a s W ell a s
thos e pa r ts ther eof W hich ha v e f unctiona l cons ens us or
clea v a g e s ite or binding dom a in s eq uence( s ) neces s a r y f or a
des ir ed pr otein to inter a ct W ith the nucleic a cid a ccor ding to
the inv ention.
N ov . 22, 2001
[ 0014] For ex a m ple, the pr es ent inv ention pa r ticula r ly
pr ov ides a n im pr ov ed m ethod f or tr a ns iently tr a ns f ecting /
tr a ns f or m ing or f or s ta bly tr a ns f or m ing a nim a l cells W ith
ex og enous nucleic a cids s uch a s eg D N A, W hich com bines
pos itiv e a ttr ibutes of Ag r oba cter ium tum ef a ciens m edia ted
T- D N A tr a ns f er a nd integ r a tion W ith non- biolog ica l deliv er y
m ethods . This a s pect of the inv ention com pr is es pr ov iding
a n a nim a l cell W ith the ex og enous D N A f r a g m ent des ir ed to
be integ r a ted into the a nim a l cell g enom e, bounded by
T- D N A bor der s or f unctiona l pa r ts ther eof , a long W ith a t
lea s t one Ag r oba cter ium - der iv ed pr otein tha t pr om otes the
integ r a tion of the ex og enous D N A into the hos t cell g enom e.
The Ag r oba cter ium - der iv ed pr otein us ed a ccor ding to the
inv ention pa r ticula r ly includes Vir D l, Vir D 2, Vir E2, a nd
Vir C. Pr ef er a bly , a com bina tion of Vir D 2 a nd either Vir D l,
Vir C, Vir E2, or a s ubcom bina tion ther eof , is us ed. Mos t
pr ef er a bly , us e is m a de of the Ag r oba cter ium - der iv ed pr o
teins Vir D 2 a nd Vir E2 in com bina tion. HoW ev er , in cer ta in
ca s es s ole us e of Vir D 2 m a y be s uf f icient a nd is a ls o W ithin
the s cope of the inv ention.
[ 0015] Accor ding to the pr es ent inv ention it ha s s ur pr is
ing ly been f ound tha t a nim a l cells der iv ed f r om - v a r ious cell
lines a r e s ucceptible f or tr a ns f or m a tion by us ing the D N A/
pr otein com plex a s dis clos ed in W O 9 5/ 0547 1 m entioned
a bov e, a nd ca n ef f iciently be tr a ns iently tr a ns f ected by us ing
a s im ila r com plex in W hich the nucleic a cid com ponent is in
the f or m of a n olig onucleotide ena bling a ntis ens e- , a ntig ene
a nd olig oZy m e- a ppr oa ches . Fur ther m or e, it ha s been f ound
tha t the Ag r oba cter ium der iv ed v ir ulence pr oteins , Vir D l
a nd Vir D 2, inter a ct W hen ex pr es s ed in m a m m a lia n cells . In
pa r ticula r , the a bov e m a in obj ect under ly ing the pr es ent
inv ention ha s thus been- a chiev ed by us ing the pr oper ties of
pr oteins pr oduced by the v ir r eg ion of Ag r oba cter ium s uch
a s , f or ex a m ple, Vir D l, Vir D 2 a nd Vir E2, but es pecia lly tha t
of the Vir D 2 pr oteins , in non- Ag r oba cter ium tr a ns f or m a tion
of a nim a l cells .
[ 0016 ] The nucleic a cid/ pr otein com plex a lr ea dy dis clos ed
in W O 9 5/ 0547 1 m a y be oba ined by ? r s t pr ov iding a
r ecom bina nt nucleic a cid cons tr uct tha t com pr is es in oper
a ble linka g e to the elem ents a lr ea dy m entioned a bov e a t
lea s t one T- D N A bor der s eq uence or f unctiona l pa r t ther eof
a s a s ubs tr a te in the Vir D 2 clea v a g e r ea ction. If the s a id
s ubs tr a te inv olv es not the W hole but only pa r t of the T- D N A
bor der s eq uence, it is to be ens ur ed tha t the s a id pa r tia l
s eq uence s till com pr is es thos e pa r ts of the T- D N A bor der
s eq uence tha t encom pa s s the r ecog nition a nd clea v a g e s ite
of the Vir D 2 pr otein.
[ 0017 ] The chim er ic r ecom bina nt nucleic a cid cons tr uct a s
des cr ibed a bov e is pr ef er a bly a s ing le s tr a nded D N A con
s tr uct. Als o com pr is ed W ithin the s cope of the inv ention is
a double- s ta nded m olecule W ith a s ing le- s tr a nded ov er ha ng
W hich is a s ubs tr a te f or Vir D 2, or a chim er ic r ecom bina nt
D N A cons tr uct neg a tiv ely s uper coiled ( f or m I) conta ining
bor der s eq uences or a t lea s t f unctiona l pa r ts ther eof a s the
pr ef er r ed s ubs tr a te f or Vir D 1Vir D 2 ca ta ly Zed clea v a g e.
Accor ding to a pr ef er r ed em bodim ent of the inv ention s a id
chim er ic r ecom bina nt nucleic a cid/ pr otein com plex f ur ther
com pr is es Vir E2 a nd/ or a ny other nucleic a cid binding
pr otein, s uch a s r ecA, W hich contr ibutes to ef f icient nuclea r
im por t a nd W hich, pr ef er a bly , is a ls o a ble to pr otect the
nucleic a cid to be tr a ns f or m ed or tr a ns f ected f r om nuclea s e
a tta ck. Pr ef er a bly , the inv ention thus pr ov ides us e of a
chim er ic r ecom bina nt nucleic a cid cons tr uct cov a lently
US 2001/ 0044150 A1
a s s ocia ted W ith Vir D 2, a nd optiona lly Vir E2, pr oteins f or the
tr a ns f or m a tion or tr a ns f ection of a nim a l cells ex v iv o, in
v itr o a nd in v iv o. In this contex t it is to be under s tood tha t
the pr oteins m entioned her einbef or e a s com ponent of the
nucleic a cid/ pr otein com plex a ccor ding to the inv ention
s ha ll a ls o m ea n olig opeptides or der iv a tiv es der iv ed ther e
f r om or f unctiona l f r a g m ents ther eof r eta ining a t lea s t one
f unctiona l cha r a cter is tic neces s a r y to a chiev e the a im s of the
pr es ent inv ention.
[ 0018] For deta ils concer ning the pr epa r a tion of nucleic
a cid/ pr otein com plex es W hich ca n be us ed a ccor ding to the
pr es ent inv ention r ef er ence is m a de to the com plete dis clo
s ur e of W O 9 5/ 0547 1. HoW ev er , it ha s to be noted tha t the
nucleic a cid/ pr otein com plex a ccor ding to the pr es ent inv en
tion pr ef er a bly com pr is es both Vir E2 a nd Vir D 2 pr oteins in
or der to a chiev e optim um deliv er y a nd/ or tr a ns f or m a tion
ef ? ciency , a lthoug h the s ole us e of the Vir D 2 pr otein in the
com plex , in pa r ticula r if s a id com plex com pr is es s ing le
s tr a nded olig onucleotides , is a ls o W ithin the s cope of the
pr es ent inv ention. W ith r es pect to the pr epa r a tion of a
com plex in W hich the nucleic a cid com ponent is in the f or m
of a n olig onucleotide ena bling a ntis ens e- , a ntig ene- or oli
g oZy m e- a ppr oa ches , it is clea r f or a per s on s killed in the a r t
hoW to cons tr uct s uch a com plex .
[ 0019 ] Upon us ing the D N A/ pr otein com plex a ccor ding to
W O 9 5/ 0547 1 f or the tr a ns f or m a tion of a nim a l cells , the
tr a ns f or m a tion f r eq uency a nd a ls o the q ua lity of the inte
g r a ted D N A ca n be im pr ov ed cons ider a bly . This is es pe
cia lly tr ue W ith r eg a r d to s ta ble tr a ns f or m a tion ev ents , W hich
occur m or e f r eq uently a s com pa r ed to conv entiona l, non
pr otein a s s ocia ted D N A cons tr ucts .
[ 0020] The pr es ent inv ention thus com pr is es a m ethod f or
intr oducing nucleic a cids into cells , eg f or tr a ns f or m ing or
tr a ns iently tr a ns f ecting / tr a ns f or m ing a nim a l cells , com pr is
ing
[ 0021] ( a ) pr epa r ing a chim er ic r ecom bina nt nucleic
a cid cons tr uct tha t com pr is es in oper a ble linka g e to
a n ex pr es s ible D N A or to a n olig onucleotide a t lea s t
one T- D N A bor der s eq uence or f unctiona l pa r t
ther eof , W hich s er v es a s a s ubs tr a te in the Vir D 2
clea v a g e r ea ction, a s de? ned her einbef or e;
[ 0022] ( b) clea v ing of the nucleic a cid s ubs tr a te pr e
pa r ed a ccor ding to s tep ( a ) by m ea ns of Vir D 2
pr otein, W hich m a y be a ccom pa nied by f ur ther Vir
pr oteins s uch a s , f or ex a m ple Vir D 1 a nd/ or Vir E2
a nd/ or a ny other nucleic a cid binding pr otein, W hich
is a ble to pr otect s a id nucleic a cid f r om nuclea s e
a tta ck;
[ 0023 ] ( c) intr oducing the thus clea v ed nucleic a cid
com pr is ing a t lea s t the Vir D 2 pr otein cov a lently
bound to the 5 end of the clea v a g e s ite into the cells
to be tr a ns f or m ed or tr a ns f ected by m ethods knoW n
in the a r t.
[ 0024] Accor ding to a pr ef er r ed em bodim ent of the a bov e
m ethod, clea v ing of the nucleic a cid s ubs tr a te ( b) is ca r r ied
out in v itr o.
[ 0025] Accor ding to the inv ention the nucleic a cid/ Vir D 2
pr otein com plex is pr ef er a bly a ccom pa nied by f ur ther Vir
pr oteins , s uch a s , f or ex a m ple, Vir E2, W hich is knoW n to
bind to s s D N A, a nd/ or Vir D 1. Vir E2 ca n be pur i? ed by
N ov . 22, 2001
m ethods knoW n in the a r t s uch a s thos e des cr ibed in Chr is tie
et a l [ J B a cter iol 17 0( 6 ) : 26 59 - 26 6 7 ( 19 88) ] . The pur i? ca
tion of the Vir D 1 pr otein ca n be a chiev ed a ccor ding to the
m ethod dis clos ed in W O 9 5/ 0547 1, W her ea s Vir D 2 ca n be
obta ined a s s et f or th in Pa ns eg r a u et a l. [ PN AS 9 0, 1153 8
( 19 9 3 ) ] .
[ 0026 ] As m entioned bef or e, the m a in obj ect of the
pr es ent inv ention is the us e of a nucleic a cid/ pr otein com
plex com pr is ing oper a bly linked to eg a n ex pr es s ible D N A
or to a n olig onucleotide a t lea s t one T- D N Abor der s eq uence
or f unctiona l pa r t ther eof , a nd cov a lently a s s ocia ted ther e
W ith a Vir D 2 pr otein in a pr oces s f or intr oducing nucleic
a cids into a nim a l cells . The nucleic a cid/ Vir D 2 pr otein
com plex pr ef er a bly conta ins non- cov a lently a s s ocia ted f ur
ther Vir pr oteins s uch a s , f or ex a m ple, Vir D 1 a nd/ or Vir E2,
W ith Vir E2 being m os t pr ef er r ed, pa r ticula r ly if us ed in
connection W ith s ing le- s tr a nded olig onucleotides .
[ 0027 ] The ter m ex og enous D N A or nucleic a cid us ed
her ein is m ea nt to include a ny D N A or other nucleic a cid
tha t ha s been obta ined by r ecom bina nt nucleic a cid tech
nolog y . The ex og enous nucleic a cid to be us ed in the pr oces s
a ccor ding to the inv ention f or tr a ns f or m ing or tr a ns f ecting
ta r g et cells m a y be either of hom olog ous or heter olog ous
or ig in W ith r es pect to the cell ty pe inv olv ed or it m a y be of
s y nthetic or ig in or both. The coding nucleic a cid s eq uence
ca n be cons tr ucted a ccor ding to conv entiona l m ethods , eg
f r om g enom ic D N A, or f r om cD N A. Another pos s ibility is
the cons tr uction of a hy br id D N A s eq uence cons is ting of
both cD N A a nd g enom ic D N A a nd/ or s y nthetic D N A. The
cD N A m a y or ig ina te f r om the s a m e g ene a s the g enom ic
D N A, or a lter na tiv ely both the cD N A a nd the g enom ic D N A
m a y or ig ina te f r om dif f er ent g enes . In a ny ca s e, hoW ev er ,
both the g enom ic D N A a nd/ or the cD N A m a y ea ch be
pr epa r ed indiv idua lly f r om the s a m e or f r om dif f er ent g enes .
[ 0028] The ter m s y nthetic D N A or nucleic a cid includes
( a ) nucleic a cid s eq uences tha t ha v e been pr epa r ed entir ely
or a t lea s t pa r tia lly by chem ica l m ea ns a nd ( b) a ntis ens e or
s ens e olig onucleotides . For ex a m ple, s y nthetic D N A
s eq uences m a y be s uita bly us ed, e. g . , f or m odif y ing na tiv e
D N A s eq uences in ter m s of codon us a g e, ex pr es s ion ef ?
ciency , etc.
[ 0029 ] Another a ppr oa ch a ccor ding to the pr es ent inv en
tion is to g ener a te a ntis ens e or a ntig ene RN A or r iboZy m es /
olig oZy m es W ithin a g iv en hos t cell by intr oducing into s a id
cell a nucleic a cid W ith r ev er s e or ienta tion W hich m a y
becom e pa r t of the hos t cells g enom e. In this contex t,
intr oducing nucleic a cid f r a g m ents encoding a ntis ens e or
a ntig ene olig onucleotides or olig oZy m es cor r es ponding to
ev en les s tha n 100 bp of a s peci? c RN A or D N A to be
ta r g eted is s uf ? cient f or inhibiting or m odula ting tr a ns la tion.
[ 003 0] If the nucleic a cid s eq uence to be tr a ns f er r ed into
the r ecipient cell conta ins por tions of m or e tha n one g ene,
thes e g enes m a y or ig ina te f r om one a nd the s a m e or g a nis m ,
f r om s ev er a l or g a nis m s tha t belong to m or e tha n one s tr a in,
one v a r iety or one s pecies of the s a m e g enus , or f r om
or g a nis m s tha t belong to m or e tha n one g enus of the s a m e
or of a nother ta x onom ic unit ( king dom ) . Chim er ic r ecom
bina nt nucleic a cid m olecules tha t com pr is e a n ex pr es s ible
D N A, but es pecia lly a s tr uctur a l g ene, pr ef er a bly a heter
olog ous s tr uctur a l g ene oper a bly linked W ith ex pr es s ion
s ig na ls a ctiv e in r ecipient cells , s uch a s enha ncer , pr om oter
a nd tr a ns cr iption ter m ina tion s eq uences , a s W ell a s , option
US 2001/ 0044150 A1
a lly , W ith f ur ther coding a nd/ or non- coding s eq uences of the
5 a nd/ or 3 r eg ion s uch a s eg s ig na l s eq uence, m a y a ls o be
pr ef er a bly us ed W ithin the tr a ns f or m a tion pr oces s a s pa r t of
the nucleic a cid/ pr otein com plex us ed a ccor ding to the
pr es ent inv ention. It is of ten a dv a nta g eous to incor por a te a
lea der s eq uence betW een the pr om oter s eq uence a nd the
a dj a cent coding D N A s eq uence, the leng th of the lea der
s eq uence being s o s elected tha t the dis ta nce betW een the
pr om oter a nd the D N A s eq uence to be ex pr es s ed is the
optim um dis ta nce f or ex pr es s ion of the a s s ocia ted s tr uctur a l
g ene.
[ 003 1] Fur ther m or e, the ex og enous D N A or other nucleic
a cid f or m ing pa r t of the nucleic a cid/ pr otein com plex m a y
a dditiona lly com pr is e s eq uences encoding one or m or e
s electa ble m a r ker s us ef ul in s cr eening f or pos itiv e tr a ns f or
m a nts . In g ener a l, thes e m a r ker s a r e pr oteins neces s a r y f or
the s ur v iv a l or g r oW th of tr a ns f or m ed hos t cells g r oW n in a
s electiv e cultur e m edium . Hos t cells not tr a ns f or m ed W ith
the v ector conta ining the s election g ene W ill not s ur v iv e in
the cultur e m edium . Ty pica l s election m a r ker s encode pr o
teins tha t conf er r es is ta nce to a ntibiotics a nd other - tox ins ,
e. g . a m picillin, hy g r om y cin, neom y cin, pur om y cin, m eth
otr ex a te or tetr a cy cline, com plem ent a ux otr ophic de? cien
cies , or s upply cr itica l nutr ients not a v a ila ble f r om com plex
m edia . Fur ther ex a m ples of g enes tha t conf er a ntibiotic
r es is ta nce include thos e coding f or the ka na m y cin r es is ta nce
( N PT II) g ene der iv ed f r om Tn5 ( B ev a n et a l, N a tur e 3 04:
184- 187 ( 19 83 ) ) , a nd chlor a m phenicol a cety ltr a ns f er a s e.
[ 003 2] Suita ble s electa ble m a r ker s f or a nim a l, pa r ticula r ly
m a m m a lia n cells a r e thos e tha t ena ble the identi? ca tion of
cells com petent to ta ke up the nucleic a cid encoding s a id
s electa ble m a r ker , s uch a s dihy dr of ola te r educta s e ( D HFR,
m ethotr ex a te r es is ta nce) , thy m idine kina s e, or g enes con
f er r ing r es is ta nce to G418 or hy g r om y cin ( s ee eg
B lochling er a nd D ig g elm a nn ( 19 84) , Molecula r a nd Cellu
la r B iolog y 4: 29 29 - 29 3 1; Rober ts on a nd W ha lley ( 19 88) ,
N ucl. Acids Res . 16 : 113 03 - 113 17 ; O B r ia n et a l. ( 19 9 7 ) ,
Gene 184: 115- 120) . The a nim a l cell tr a ns f or m a nts a r e
pla ced under s election pr es s ur e W hich only thos e tr a ns f or
m a nts W hich ha v e ta ken up a nd a r e ex pr es s ing the m a r ker
a r e uniq uely a da pted to s ur v iv e. In the ca s e of a D HFR or
g luta m ine s y ntha s e ( GS) m a r ker , s election pr es s ur e ca n be
im pos ed by cultur ing the tr a ns f or m a nts under conditions in
W hich the pr es s ur e is pr og r es s iv ely incr ea s ed, ther eby lea d
ing to a m pli? ca tion ( a t its chr om os om a l integ r a tion s ite) of
both the s election g ene a nd the linked D N A tha t encodes a
s tr uctur a l g ene of inter es t des ir ed to be ex pr es s ed in the
tr a ns f or m ed cells . Am pli? ca tion is the pr oces s by W hich
g enes in g r ea ter dem a nd f or the pr oduction of a pr otein
cr itica l f or g r oW th, tog ether W ith clos ely a s s ocia ted g enes
W hich m a y encode a des ir ed pr otein, a r e r eiter a ted in ta ndem
W ithin the chr om os om es of r ecom bina nt cells . Incr ea s ed
q ua ntities of des ir ed pr otein a r e us ua lly s y nthes is ed f r om
thus a m pli? ed D N A. For the pur pos e of s cr eening tr a ns ient
ex pr es s ion of the des ir ed g ene intr oduced into a s uita ble
a nim a l hos t cell a ccor ding to the inv ention the ex og enous
D N A m a y a ls o com pr is e s eq uences encoding [ 3 - g a la ctos i
da s e, g r een ? uor es cent pr otein ( g f p) , or lucif er a s e. Methods
f or the detection of the ex pr es s ion of s a id m a r ker s a r e W ell
knoW n in the a r t. Scr eening of a nim a l cells a nd a nim a ls
der iv ed f r om s uch cells f or the pr es ence of s peci? c nucleic
a cid s eq uences m a y a ls o be per f or m ed by Souther n a na ly s is
[ Souther n, J . Mol. B iol. 9 8: 503 ( 19 7 5) ] . D eta ils of this
pr ocedur e a r e g iv en in Ma nia tis et a l, Molecula r Cloning : A
N ov . 22, 2001
La bor a tor y Ma nua l, Cold Spr ing Ha r bor La bor a tor y , Cold
Spr ing Ha r bor , N Y. ( 1 9 89 ) . This s cr eening m a y a ls o be
per f or m ed by the us e of Poly m er a s e Cha in Rea ction pr oce
dur es ( PCR) . PCR pr ocedur es a r e des cr ibed in deta il in
Mullis et a l, Meth. EnZy m ol. 155: 3 3 5- 3 50 ( 19 87 ) a nd
Er lich, ( ed. ) , PCR Technolog y , Stockton Pr es s , N eW Yor k
( 19 89 ) .
[ 003 3 ] The ex pr es s ion s ig na ls a ctiv e in ta r g et cells us ua lly
com pr is e a pr om oter tha t is r ecog nis ed by the hos t or g a nis m
a nd is oper a bly linked to the D N A to be ex pr es s ed in the
tr a ns f or m a nt. Such a pr om oter m a y be inducible or cons ti
tutiv e. The pr om oter s a r e oper a bly linked to s a id D N A by
r em ov ing the pr om oter f r om the s our ce D N A by r es tr iction
enZy m e dig es tion a nd com bining the is ola ted pr om oter
s eq uence W ith the ex pr es s ible D N A s eq uence. B oth the
na tiv e pr om oter s eq uence of the s tr uctur a l g ene of inter es t
a nd m a ny heter olog ous pr om oter s m a y be us ed to dir ect
a m pli? ca tion a nd/ or ex pr es s ion of s a id s tr uctur a l g ene.
Suita ble pr om oter s f or a nim a l a nd in pa r ticula r m a m m a lia n
hos ts a r e thos e der iv ed f r om the g enom es of v ir us es s uch a s
poly om a v ir us , a denov ir us , f oW lpox v ir us , bov ine pa pillom a
v ir us , a v ia n s a r com a v ir us , Rous e s a r com a v ir us ( RSV) ,
cy tom eg a lov ir us ( CMV) , a r etr ov ir us a nd Sim ia n Vir us 40
( SV40) , f r om heter olog ous m a m m a lia n pr om oter s s uch a s
the [ 3 - a ctin pr om oter or a v er y s tr ong pr om oter , eg a
r ibos om a l pr otein pr om oter , a nd f r om the pr om oter nor m a lly
a s s ocia ted W ith s tr uctur a l g ene s eq uence to be ex pr es s ed,
pr ov ided s uch pr om oter s a r e com pa tible W ith the hos t cell
s y s tem s .
[ 003 4] The tr a ns cr iption of a n ex og enous D N A encoding
the des ir ed s tr uctur a l g ene ca n- be incr ea s ed by ins er ting a n
enha ncer s eq uence into the D N A a s a com ponent of the
nucleic a cid/ pr otein com plex a ccor ding to the inv ention.
Enha ncer s a r e r ela tiv ely or ienta tion a nd pos ition indepen
dent. Ma ny enha ncer s eq uences a r e knoW n f r om m a m m a lia n
g enes ( e. g . ela s ta s e a nd g lobin) . HoW ev er , ty pica lly one W ill
em ploy a n enha ncer f r om a euka r y otic cell v ir us . Ex a m ples
include the SV40 enha ncer on the la te s ide of the r eplica tion
or ig in ( bp 100- 27 0) a nd the CMV ea r ly pr om oter enha ncer .
The enha ncer m a y be s pliced into the r ecom bina nt chim er ic
s eq uence a t a pos ition 5 or 3 to the coding D N A s eq uence,
but is pr ef er a bly loca ted a t a s ite 5 f r om the pr om oter .
[ 003 5] Hos t cells to W hich nucleic a cids ca n be deliv er ed
by a m ethod a ccor ding to the inv ention include ins ect a nd
v er tebr a te cells . In r ecent y ea r s pr opa g a tion of v er tebr a te
cells in cultur e ( tis s ue cultur e) ha s becom e a r outine pr oce
dur e. Ex a m ples of us ef ul v er tebr a te hos t cell lines a r e
epithelia l or ? br obla s tic cell lines s uch a s Chines e ha m s ter
ov a r y ( CHO) cells , COS1 cells ( m onkey kidney cells tr a ns
f or m ed W ith SV40 T- a ntig en) , CV1 cells ( pa r ent line of the
f or m er ) , Ra t1 ( r a t ? br obla s t) cells , N IH 3 T3 cells , HeLa
cells , LLC- Pk1 ( pig kidney epithelia l) cells or 29 3 T cells .
The hos t cells r ef er r ed to in this dis clos ur e com pr is e cells in
in v itr o/ ex v iv o cultur e a s W ell a s cells tha t a r e W ithin a hos t
a nim a l.
[ 003 6 ] Accor ding to a f ur ther a s pect of the pr es ent inv en
tion r ela ting to a ntis ens e- , a ntig ene- or olig oZy m e- a p
pr oa ches , the g r oup of hos t cells W hich ca n be ta r g eted a ls o
includes pla nt cells or tis s ues , W hich pr ef er a bly ca n be
r eg ener a ted to W hole pla nts .
[ 003 7 ] Es pecia lly s uita ble f or us e in the pr oces s a ccor ding
to the inv ention a r e a ll thos e s tr uctur a l g enes W hich upon
US 2001/ 0044150 A1
ex pr es s ion pr oduce pr oteins or poly peptides W hich a r e ben
e? cia l f or the tr a ns f or m ed cells , tis s ues or a nim a ls , eg
W hich com pens a te ev entua l m uta ta tions , or W hich ha v e
pha r m a colog ica l pr oper ties a nd could be us ed a s pha r m a
ceutica l a g ents in the tr ea tm ent of dis ea s es . Ex a m ples f or
s uch s tr uctur a l g enes include thos e encoding hor m ones ,
im m unom odula tor s a nd other phy s iolog ica lly a ctiv e s ub
s ta nces .
[ 003 8] The g enes tha t pa r ticula r ly com e into cons ider a tion
W ithin the s cope of this inv ention ther ef or e include, but a r e
not lim ited to, f or ex a m ple, m a m m a l- s peci? c g enes , s uch a s
the ins ulin g ene, the s om a tos ta tin g ene, the inter leukin
g enes , the t- PA g ene, etc. , or g enes of m icr obia l or ig in, s uch
a s the N PT II g ene, etc. a nd s y nthetic g enes , s uch a s the
ins ulin g ene, etc.
[ 003 9 ] Apa r t f r om na tur a lly occur r ing s tr uctur a l g enes
tha t code f or a us ef ul a nd des ir a ble pr oper ty or a pha r m a
colog ica l a g ent, W ithin the s cope of this inv ention it is a ls o
pos s ible to us e g enes tha t ha v e been m odi? ed pr ev ious ly in
a s peci? c m a nner us ing chem ica l or g enetic eng ineer ing
m ethods .
[ 0040] Fur ther m or e, the br oa d concept of the pr es ent
inv ention a ls o includes g enes tha t a r e pr oduced entir ely or
pa r tia lly by chem ica l s y nthes is . Genes or D N A s eq uences
tha t m a y be us ed W ithin the s cope of the pr es ent inv ention
a r e ther ef or e both hom olog ous a nd heter olog ous g ene( s ) or
D N A a nd a ls o s y nthetic g ene( s ) or D N A a ccor ding to the
de? nition g iv en W ithin the s cope of the pr es ent inv ention.
The ins ulin g ene m a y be m entioned a t this point a s a n
ex a m ple of a s y nthetic g ene.
[ 0041] Alter na tiv ely , olig onucleotides ca n be us ed cor r e
s ponding in s eq uence to a cellula r s eq uence to be ta r g eted,
either in the s a m e ( a ntig ene) coding dir ection, a s s uch or
ca r r y ing a m uta tion, or in the a ntis ens e coding dir ection.
[ 0042] Pos s ible m ethods f or the dir ect tr a ns f er of the
nucleic a cid/ pr otein com plex a ccor ding to the inv ention into
a cell com pr is e, f or ex a m ple, the tr ea tm ent of cells us ing
pr ocedur es tha t m odif y the pla s m a m em br a ne, f or ex a m ple,
poly ethy lene g ly col tr ea tm ent, lipos om e- ba s ed technolo
g ies , hea t s hock tr ea tm ent or electr opor a tion, or a com bi
na tion of thos e pr ocedur es ( s ee eg Chu et a l. ( 19 87 ) , N ucl.
Acids Res . 15: 13 11- 13 26 ; Hodg s on a nd Sola im a n ( 19 9 6 ) ,
N a tur e B iotech. 14: 3 3 9 - 3 42; Shillito et a l. ( 19 85) , B io
Technolog y 3 : 109 9 - 1103 ) .
[ 0043 ] In the electr opor a tion techniq ue, cells tog ether W ith
the nucleic a cid/ pr otein com plex us ed a ccor ding to the
inv ention a r e s ubj ected to electr ica l puls es of hig h ? eld
s tr eng th. This r es ults in a r ev er s ible incr ea s e in the per m e
a bility of biom em br a nes a nd thus a lloW s the ins er tion of the
nucleic a cid/ pr otein com plex a ccor ding to the inv ention.
Electr opor a ted cells r eneW their cell m em br a ne, div ide a nd
f or m a g g r eg a tes or m onola y er s of tr a ns f or m ed cells . Selec
tion of the tr a ns f or m ed cells ca n ta ke pla ce W ith the a id of
the a bov e- des cr ibed phenoty pic m a r ker s .
[ 0044] A f ur ther m ethod f or the dir ect intr oduction of the
nucleic a cid/ pr otein com plex us ed a ccor ding to the inv en
tion into cells , W hich is ba s ed on pur ely chem ica l pr ocedur es
a nd W hich ena bles the tr a ns f or m a tion to be ca r r ied out v er y
ef ? ciently a nd r a pidly , is des cr ibed in J or da n et a l. ( 19 9 6 ) ,
N ucl. Acids Res . 24: 59 6 - 6 01) .
N ov . 22, 2001
[ 0045] Als o s uita ble f or the tr a ns f or m a tion of eg a nim a l
cells is dir ect g ene tr a ns f er us ing co- tr a ns f or m a tion
[ Schocher RJ et a l, B io/ Technolog y , 4: 109 3 - 109 6 ( 19 86 ) ] .
Co- tr a ns f or m a tion is a m ethod tha t is ba s ed on the s im ul
ta neous ta king up a nd integ r a tion of v a r ious D N A m olecules
( non- s electa ble a nd s electa ble g enes ) into the r ecipient s
g enom e a nd tha t ther ef or e a lloW s the detection of cells tha t
ha v e been tr a ns f or m ed W ith non- s electa ble g enes .
[ 0046 ] Fur ther m ea ns f or ins er ting the nucleic a cid/ pr otein
com plex us ed a ccor ding to the inv ention dir ectly into a cell
com pr is e us ing pur ely phy s ica l pr ocedur es , f or ex a m ple by
m icr oinj ection us ing ? nely dr a W n m icr opipettes [ N euha us et
a l ( 19 87 ) ] or by bom ba r ding the cells W ith m icr opr oj ectiles
tha t a r e coa ted W ith the tr a ns f or m ing or tr a ns iently tr a ns
f ecting nucleic a cid [ Micr opr oj ectile B om ba r dm ent ;
W a ng Y- C et a l, Pla nt Mol. B iol. 11: 43 3 - 43 9 ( 19 88) ] or a r e
a cceler a ted thr oug h a nucleic a cid conta ining s olution in the
dir ection of the cells to be tr a ns f or m ed by a pr es s ur e im pa ct
ther eby being ? nely a tom iZed into a f og W ith the s olution a s
a r es ult of the pr es s ur e im pa ct [ EP- A- 43 4, 6 16 ] . Micr o
pr oj ectile bom ba r dm ent ha s been a dv a nced a s a n ef f ectiv e
tr a ns f or m a tion techniq ue f or eg a nim a l cells .
[ 0047 ] The lis t of pos s ible tr a ns f or m a tion a nd tr a ns f ection
m ethods g iv en a bov e by W a y of ex a m ple is not cla im ed to
be com plete a nd is not intended to lim it the s ubj ect of the
inv ention in a ny W a y .
[ 0048] The pr es ent inv ention a ls o concer ns the pr epa r a
tion of tr a ns g enic a nim a l cells , including oocy tes , s per m a
tocy tes a nd Zy g otes etc. , tr a ns g enic or g a ns a nd tr a ns g enic
a nim a ls , a s W ell a s the cells a nd a nim a ls obta ined by us e of
a m ethod a ccor ding to the inv ention.
[ 0049 ] A tr a ns g enic a nim a l W hich ca n be pr oduced
a ccor ding to the inv ention pr ef er a bly is a m a m m a l, W ith
pig s , r odents a nd r um ina nts being m os t pr ef er r ed. Addition
a lly , the pr es ent inv ention ca n be us ed f or s om a tic g ene
ther a py in hum a ns , W hich us e is a ls o pa r t of the inv ention.
[ 0050] The m ethod a ccor ding to the inv ention ca n be
a dv a nta g eous ly us ed to incr ea s e The tr a ns f or m a tion ef ?
ciency of non- Ag r oba cter ium m edia ted tr a ns f or m a tion pr o
ces s es , in tha t, f or ex a m ple, les s tr a ns f or m ing D N A is
needed a s com pa r ed to the conv entiona l techniq ues . In
a ddition the q ua lity of the integ r a ted D N A ca n be im pr ov ed
by the pr ecis ion of the integ r a tion pr oces s , a nd pos s ible
r ea r r a ng em ents W hich a r e likely to ha ppen to na ked D N A
ca n be a v oided.
[ 0051] The m ethod a ccor ding to the inv ention thus pr o
v ides v a lua ble m ea ns f or the tr ea tm ent of v a r ious dis or der s
s ucceptible to g ene ther a py a nd ena bles the pr oduction of
tr a ns g enic a nim a ls , W her e the ef f iciency of integ r a tion of
na ked D N A is a lim iting f a ctor . Fur ther m or e, the m ethod is
us ef ul in the tr ea tm ent of ca ncer cells , a s a neW non- v ir a l
s y s tem W ithout LTR a nd pos s ible ha Za r ds connected W ith
them . A s pecia l f ea tur e of the com plex es us ed a ccor ding to
the inv ention is their D N As e r es is ta nce a nd their a bility to
a ls o ta r g et non div iding cells , due to their nuclea r ta r g eting
potentia l.
[ 0052] The inv ention is f ur ther des cr ibed, f or the pur pos es
of illus tr a tion only , in the f olloW ing ex a m ples .
US 2001/ 0044150 A1
EXAMPLES
Ex a m ple 1
Cons tr uction of Pla s m ids f or Monitor ing the
Intr a cellula r Loca lis a tion of Vir D 2 Pr otein
[ 0053 ] The N ter m ina l g f p f us ion v ector p[ 3 a ct- N GFP,
conta ining the [ 3 - a ctin pr om oter a nd SV40 ter m ina tor is us ed
( Ludin et a l. ( 19 9 6 ) Gene 17 3 : 107 - 111) . v ir D 2 is cloned a s
entir e g ene, or a s a m uta nt g ene conta ining only the N - ter
m ina l ( Ros s i et a l. ( 19 9 3 ) Mol. Gen Genet. 23 9 : 3 45- 3 53 ) ,
or C ter m ina l N LS, or m uta nt g ene in W hich both nuclea r
loca lis a tion ( N LS) s eq uences a r e deleted. For detection of
the pr otein by a nti- Vir D 2 a ntibodies , v ir D 2 is cloned in
m a m m a lia n ex pr es s ion v ector pcD N A3 ( Inv itr og en) .
Ex a m ple 2
Cons tr uction of Pla s m id f or Monitor ing the
Intr a cellula r Loca lis a tion of Vir D 1 Pr otein
[ 0054] Vir D 1 g ene is a m pli? ed by PCR us ing pVCK225
( V. C. Kna uf a nd E. W . N es ter , Pla s m id 8, 45- 54 ( 19 82) ) a s
a tem pla te. The hea m a g g lutinin ( HA) epitope ta g g ed con
s tr uct pHA- D 1 is pr epa r ed by lig a ting a n HA epitope
encoding olig onucleotide to the 5 end of the v ir D 1 PCR
pr oduct, in- f r a m e W ith the initia tor m ethionin codon, in the
m a m m a lia n ex pr es s ion v ector pcD N A3 ( Inv itr og en) .
Ex a m ple 3
Monitor ing Intr a cellula r Loca liza tion of Vir D 2
Pr otein in Ma m m a lia n Cells
[ 0055] W hen ov er ex pr es s ed in m a m m a lia n cells ( HeLa ,
29 3 ) , Vir D 2 pr otein s hoW s ex clus iv ely nuclea r loca lis a tion,
W hich is m onitor ed either by GFP- Vir D 2 f us ion, or by
im m unor ea ction of v ir D 2 tr a ns f ected cells W ith a nti- Vir D 2
a ntibodies . The pr es ence of a ny of tW o loca lis a tion s ig na ls ,
on the N - a nd C- ter m inus of the Vir D 2, is s uf ? cient f or
ef ? cient nuclea r loca lis a tion, W hile deletion of both N LS
s eq uences r ender s the pr otein cy topla s m ic. Ov er ex pr es s ion
of Vir D 2 pr otein in m a m m a lia n cells does not ha v e a v is ible
neg a tiv e ef f ect on their g r oW th a nd div is ion.
Ex a m ple 4
Monitor ing Intr a cellula r Loca lis a tion of Vir D 1
Pr otein in Ma m m a lia n Cells
[ 0056 ] W hen ov er ex pr es s ed in m a m m a lia n cells ( HeLa ,
29 3 ) , Vir D 1 pr otein s hoW s ex clus iv ely cy topla s m ic loca li
s a tion W hich is m onitor ed by im m unor ea ction of pHA- D 1
tr a ns f ected cells W ith a n a nti- HA epitope 12CA5 m ono
clona l a ntibody ( B oehr ing er ) . Ov er ex pr es s ion of Vir D 1 pr o
tein in m a m m a lia n cells does not ha v e a v is ible neg a tiv e
ef f ect on their g r oW th a nd div is ion.
Ex a m ple 5
Pr oduction of M13 s s D N A f or Ana ly s is of N uclea r
Ta r g eting by Gene Ex pr es s ion
[ 0057 ] Fir s t, the g f p cy cle3 g ene is cloned f r om potGFP
cy cle3 v ector ( Cr a m er i et a l. ( 19 9 6 ) N a tur e B iotechnolog y
14: 3 15- 3 19 ) into the Sm a l s ite of pB lues cr ipt SKII a s Stul
f r a g m ent. Stul ends a r e blunted W ith T4 D N A poly m er a s e.
Then, g f pcy cle3 g ene is cloned a s N otl/ Ps tl f r a g m ent in the
cor es ponding s ites of M13 v ector conta ining the r ig ht bor der
N ov . 22, 2001
s eq uence, na m ed Y3 ( M13 RB MCS) . Pha g e inf ection is
done in E. coli N M522. B a cter ia a r e g r oW n f or 5h a t 3 7 C.
a nd s s D N A is ola ted f r om the s uper na ta nt by Q ia g en pla s m id
pur i? ca tion kit.
Ex a m ple 6
Pr oduction of the Com plex es Us ed in D ir ect As s a y
f or Pr otein Im por t a nd Micr oinj ection
[ 0058] D N A is ? uor es cently la beled by intr oducing
r hoda m ine dUTP into the PCR r ea ction pr oduct. The pr im
er s us ed f or the PCR both conta in the r ig ht bor der ( RB )
s eq uence a t ea ch ex tr em ity of D N A, in oppos ite or ienta tion.
The PCR pr oduct is then hea t dena tur ed a nd the r es ulting
s s D N A of 1 kb leng th r ea cted W ith Vir D 2 pr otein f or 1 h a t
3 7 C. The r ea ction is s topped on ice a nd a nd incuba ted on
ice W ith Vir E2 pr otein, f or a nother 3 0 .
Ex a m ple 7
Micr oinj ection of the T- D N A Com plex es into
Ma m m a lia n Cells
[ 0059 ] T- D N A com plex es , pr oduced in the s a m e W a y a s
ex pla ined a bov e, a r e m icr oinj ected into the cy topla s m of
m a m m a lia n cells ( HeLa ) a nd nuclea r ta r g eting is m onitor ed.
Since loW a m ounts of s s D N A a r e us ed in this a s s a y , intens ity
of the s ig na l ha s to be incr ea s ed by a nti- r hoda m ine a ntibod
ies . N uclea r ta r g eting is a ls o being m onitor ed by us ing a n
a ctiv e g ene pr es ent on s s D N A. The g r een- ? uor es cent pr o
tein g ene ( g f p) is cloned in a M13 v ector conta ining the r ig ht
bor der s eq uence. ( Function of the g f p is tes ted by m icr oin
j ection of both ds , a nd s s D N A into the nucleus of HeLa
cells . ) Pha g e s s D N A is pr oces s ed W ith Vir D 2, a nd Vir E2 is
a dded. Com plex es a r e m icr oinj ected in the cy topla s m of
HeLa cells a nd ex pr es s ion of GFP is m onitor ed a f ter 12- 24h.
Ex a m ple 8
Sta ble Integ r a tion of T- D N A D er iv ed f r om the
Ar ti? cia l Com plex es
[ 006 0] For tes ting the a bility of a r ti? cia l com plex es to
integ r a te T- D N A in the m a m m a lia n g enom e, a hy g r om y cin
r epor ter g ene is cloned in a n M13 v ector conta ining the r ig ht
bor der s eq uence. Alter na tiv ely , D N A is pr oduced by PCR, in
W hich ea ch of the pr im er s conta in the r ig ht bor der s eq uence.
s s D N A is com plex ed W ith Vir D 2 a nd Vir E2 pr oteins , a nd
inj ected in the cy topla s m of HeLa cells . Af ter s election
r es is ta nt clones a r e picked a nd their D N A a na ly s ed f or the
pa tter n of integ r a tion.
Ex a m ple 9
N uclea r Ta r g eting of the T- D N A Com plex es
[ 006 1] Ar ti? cia l com plex es , cons is ting of Vir D 2 pr otein
cov a lently a tta ched to the s ing le s tr a nded D N A, a nd Vir E2
pr otein, a r e tes ted in dir ect a s s a y of pr otein im por t into HeLa
nuclei. s s D N A is r hoda m ine la belled by PCR a nd dig itonin
per m ea bilis ed HeLa nuclei a r e us ed a s a ta r g et ( Ada m et a l.
( 19 9 0) J our na l of Cell B iolog y 111: 807 - 816 ) . Indeed, the
T- D N A com plex is ef ? ciently ta r g eted to the HeLa nuclei.
Ef ? cient ta r g eting is dependent on the f unction of nuclea r
loca lis a tion s ig na l of Vir D 2.
Ex a m ple 10
Ana ly s is of Vir D 2- Vir D 2 a nd Vir D 1 - Vir D 2
Inter a ctions in Ma m m a lia n Cells
[ 006 2] Pr otein- pr otein inter a ctions a r e v er i? ed in m a m
m a lia n s y s tem by s tudies of s ubcellula r loca lis a tion of
US 2001/ 0044150 A1
Vir D 1, Vir D 2 a nd its der iv a tiv e deleted in both N LSs in
HeLa a nd HEK29 3 cells us ing GFP- Vir D 2 f us ion f or loca li
s a tion of Vir D 2 a nd HA epitope f or loca lis a tion of Vir D 1.
Vir D 2 pr otein loca lis es ex clus iv ely in the nuclei W hen
ex pr es s ed in m a m m a lia n cells . D eletion of both N LS
s eq uences r ender s the pr otein cy topla s m ic. HoW ev er , this
double m uta nt is tr a ns loca ted to the nucleus in the pr es ence
of W ild ty pe Vir D 2 pr otein, indica ting Vir D 2- Vir D 2 inter
a ction in m a m m a lia n cells . Als o the Vir D 1 pr otein, by its elf
loca lis ing in the cy topla s m , m ov es to the nucleus W hen
co- ex pr es s ed W ith the W ild ty pe Vir D 2 pr otein, indica ting
Vir D 1- Vir D 2 inter a ction in m a m m a lia n cells .
Ex a m ple 11
Pur i? ca tion of the Vir D 2 Pr otein
[ 006 3 ] A s eq uence ta g encoding s ix his tidine r es idues is
a dded to the C- ter m ina l of the Vir D 2 pr otein. The r ecom
bina nt pr otein is ex pr es s ed in E. coli B L21 a nd pur i? ed by
his tidine- nickel a f ? nity chr om a tog r a phy f olloW ed by g el
? ltr a tion a nd hepa r in a f ? nity chr om a tog r a phy a ccor ding to
s ta nda r d m ethods knoW n in the a r t.
Ex a m ple 12
Pr oduction of Sing le Str a nded Vir D 2 Pr oces s ing
Subs tr a tes Conta ining the Hy g r om y cin Res is ta nce
Gene
[ 006 4] Fir s t, the hy g r om y cin r es is ta nce g ene conta ining
the D r a l/ Fs pl r es tr iction f r a g m ent of pTK- Hy g ( Clonetech)
is cloned into the Hincl s ite of the Y3 v ector ( de? ned in
ex a m ple 5) . The r ecom bina nt pla s m id is tr a ns f or m ed into
E. c0li N M522 a nd s s pha g e D N A is is ola ted. Olig onucle
otides com plem enta r y to the EcoRV a nd Ka s i s ites in the
pha g e D N A a r e us ed to m edia te the clea v a g e of the s s D N A
by EcoRV a nd Ka s I r es tr iction nuclea s es . The obta ined
EcoRV/ Ka s i s s D N A f r a g m ent conta ins the hy g r om y cin
r es is ta nce g ene pos itioned doW ns tr ea m of the r ig ht bor der
s eq uence.
Ex a m ple 13
Tr a ns f ecion of Ar ti? cia l T- D N A Com plex es into
HeLa Cells
[ 006 5] The s s D N A EcoRV/ Ka s I f r a g m ent is r ea cted W ith
either Vir E2 or Vir D 2 pr oteins a lone or is ? r s t r ea cted W ith
v ir D 2 f olloW ed by r ea ction W ith v ir E2. The r es ulting pr o
tein: s s D N A com plex es a s W ell a s unr ea cted s s D N A a r e
tr a ns f ected into HeLa cells us ing Eug ene- 6 tr a ns f ection
r ea g ent ( B oehr ing er - Ma nnheim ) . Hy g r om y cin r es is ta nt
clones a r e s elected in tW o independent ex per im ents . The
num ber of hy g r om y cin r es is ta nt clones obta ined is s ig ni?
ca ntly hig her f or cells tr a ns f ected W ith s s D N A+Vir D 2 a nd
s s D N A+Vir D 2+Vir E2 com plex es ( ta ble 1) s ug g es ting tha t
the pr oteins f a cilita te s ta ble integ r a tion of the hy g r om y cin
r es is ta nce g ene by either pr otecting the s s D N A f r om deg
r a da tion by hos t cells nuclea s es a nd/ or by f a cilita ting the
nuclea r im por t of the com plex . Tr a ns g ene copy num ber a nd
tr a ns g ene integ r ity is a na ly Zed f or s ev er a l lines f r om ex per i
m ent 1. Pr elim ina r y da ta indica te tha t the hy g r om y cin g ene
integ r a tes a t s ing le dis tinct loci in a ll line a na ly Zed s o f a r .
Pr elim ina r y r es ults a ls o indica te tha t both Vir E2 a nd Vir D 2
N ov . 22, 2001
pr oteins pr otect the s s D N A pr ior to its integ r a tion into the
g enom e. As ex pected the pr otectiv e a ction of Vir E2 s eem s to
r es ult f r om the pr otein coa ting the entir e leng th of the
s s D N A, W her ea s the cov a lent a tta chm ent of the Vir D 2
pr otein to the 5 end of the T- D N A s peci? ca lly pr otects the
5 end.
TAB LE 1
N um ber of hy g r om y cin r es is ta nt clones obta ined
in tr a ns f ection ex per im ents
Tr a ns f ected D N A Res is ta nt clones Res is ta nt clones
( com plex ) Ex per im ent 1 Ex per im ent 2
s s D N A 6 11
s s D N A + Vir EZ 6 29
s s D N A + Vir D Z 7 3 4
s s D N A + Vir D Z + Vir EZ 12 44
1. A m ethod f or intr oducing nucleic a cids into cells ,
com pr is ing :
( a ) pr epa r ing a chim er ic r ecom bina nt nucleic a cid con
s tr uct tha t com pr is es in oper a ble linka g e to a n ex pr es s
ible D N A or to a n olig onucleotide a t lea s t one T- D N A
bor der s eq uence or f unctiona l pa r t ther eof , W hich
s er v es a s a s ubs tr a te in the Vir D 2 clea v a g e r ea ction, a s
de? ned her einbef or e;
( b) clea v ing of the nucleic a cid s ubs tr a te pr epa r ed a ccor d
ing to s tep ( a ) by m ea ns of Vir D 2 pr otein, W hich m a y
be a ccom pa nied by f ur ther Vir pr oteins s uch a s , f or
ex a m ple Vir D 1 a nd/ or Vir E2 a nd/ or a ny other nucleic
a cid binding pr otein, W hich is a ble to pr otect s a id
nucleic a cid f r om nuclea s e a tta ck;
( c) intr oducing the thus clea v ed nucleic a cid com pr is ing
a t lea s t the Vir D 2 pr otein cov a lently bound to the 5 end
of the clea v a g e s ite into the cells to be tr a ns f or m ed by
m ethods knoW n in the a r t.
2. A m ethod a ccor ding to cla im 1, W her ein the tr a ns f or
m a tion or tr a ns f ection is a chiev ed by a m ethod s elected f r om
the g r oup cons is ting of m icr oinj ection, electr opor a tion of
cells , dir ect g ene tr a ns f er a nd ba llis tic pa r ticle a cceler a tion.
3 . Us e of a s ubs ta ntia lly pur e nucleic a cid/ pr otein com
plex com pr is ing a chim er ic r ecom bina nt nucleic a cid con
s tr uct cov a lently a s s ocia ted W ith a Vir D 2 pr otein obta ina ble
by the m ethod a ccor ding to cla im 1 f or the tr a ns f or m a tion or
tr a ns f ection of a nim a l cells .
4. Us e a ccor ding to cla im 3 , W her ein the chim er ic nucleic
a cid cons tr uct com pr is es a n ex pr es s ible D N A s eq uence
under the contr ol of a nim a l or v ir a l ex pr es s ion s ig na ls .
5. Us e a ccor ding to cla im 4, W her ein the s a id ex pr es s ible
D N A s eq uence encodes f or a s tr uctur a l g ene.
6 . Us e a ccor ding to cla im 4, W her ein the s a id a nim a l
ex pr es s ion s ig na ls a r e pr om oter a nd ter m ina tion s eq uences
f unctiona l in a nim a l cells .
7 . Us e a ccor ding to cla im 3 , W her ein the chim er ic nucleic
a cid cons tr uct includes a n olig onucleotide, f or a ntis ens e- ,
a ntig ene- or olig oZy m e- a ppr oa ches .
* * * * *