Animal Cell Biotechnology: Methods and Protocols

299 Pages·1999·5.654 MB·English

by  Michelle F. Scott, Catherine M. Bentley, Carolynne T. Marshall (auth.), Nigel Jenkins (eds.)

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Preface
n1 recent year5 mamma11an 11ec 610techn0109y ha5 expanded 9reat1y fr0m
a y1ev1ta1er det1m11 6a5e, ma1n1y c0ncerned w1th vacc1ne pr0duct10n, t0 a maj0r
50urce 0f 610pharmaceut1ca15, wh1ch n0w acc0unt5 f0r 0ver 40% 0f the t0ta1
rec0m61nant 610pharmaceut1ca1 market and 2% 0f t0ta1 pharmaceut1ca1 .5e1a5
Examp1e5 0f pr0duct5 that are u5ed y11ac1n11c are m0n0c10na1 ant160d1e5; vac-
c1ne5; dru95 f0r card10va5cu1ar, re5p1rat0ry, and 1mmune d15ea5e5; and 5evera1
ant1cancer a9ent5. A1th0u9h thec urrent 5t0ck 0frec0m61nant pr0te1n5 pr0duced
n1 an1ma1 511ec that have made t1 t0 the c1n11c 51 111t5 y1ev1ta1er det1m11 (6etween
25 and 30), many m0re are n1 the en11ep1p and 111w und0u6ted1y p1ay an 9n15aercn1
r01e n1 the arm0ry 0ftherap1e5 n1 the future. n1 add1t10n t0 pr0duc1n9 rec0m61nant
pr0te1n5, 1am1na 511ec are y19n15aercn1 6e1n9 u5ed 5a a treatment n1 them5e1ve5, f0r
examp1e, n1 ex v1v0 therapy, hemat0p01e515, and 0ther f0rm5 0f 11ec rep1acement.
W1th thea dvent 0f9ene therapy ,51a1rt 1am1na 511ec are 051a u5ed 5a vect0r5 f0r the
1ntr0duct10n 0f therapeut1c 9ene5.
7he techn0109y 6eh1nd the5e new pr0duct5 can 6e very ,de211a1cep5 and d1f-
fer5 y1tnac1f1n915 fr0m the techn14ue5 u5ed n1 e1ac5-11am5 11ec cu1ture 6y 5t51tne1c5
5tudy1n9 fundamenta1 11ec .y901016 7here 51 a thu5 need f0r a v01ume 0n An1ma1
11eC 810techn0109y: Meth0d5 and Pr0t0c015, wh1ch 6r1n95 t09ether the d1ver5e
techn14ue5 u5ed n1 60th 1ndu5try and re5earch 5e1r0tar06a1 that are f0cu5ed 0n the
expre5510n 0f rec0m61nant pr0te1n5 and 0ther c0mmerc1a1 u5e5 0f 1am1na .511ec
An1ma1 11eC 810techn0109y: Meth0d5 and Pr0t0c015 51 1nt0 d1v1ded part5
that ref1ect the pr0ce55e5 re4u1red f0r d1fferent 5ta9e5 0f pr0duct10n. n1 Part 1
the 6a51c cu1ture 5e1t111caf and meth0d5 f0r the 5ucce55fu1 n01ta1t1n1 and pr0pa-
9at10n 0fce11 5en11 are de5cr16ed. n1 Chapter ,1 empha515 51 p1aced 0n pr0v1d-
9n1 m0du1ar cu1ture 5e1t111caf w1th the yt11161xe1f f0r mu1t1p1e purp05e5 and
11ec type5. 155ue5 0f 600d La60rat0ry Pract1ce (6LP) c0mp11ance and 0pera-
t0r 5afety are a150 addre55ed.
Meth0d01091e5 f0r 11ec adaptat10n [pr1nc1pa11y Ch1ne5e ham5ter 0vary
(CH0) ]511ec t0 60th 5u5pen510n and 5erum-free cu1ture are de5cr16ed n1 Chapter
2.1n add1t10n, meth0d5 f0r the cruc1a1 adaptat10n t0 h19h-den51ty cu1ture5 are
5ummar12ed (1nc1ud1n9 deve10pment 0f 11ec t01erance t0 5uch 1nh161t0ry 5u6-
5tance5 5a 1actate and amm0n1a). A1th0u9h many 0fthe f0rmu1at10n5 current1y
1v Preface
u5ed n1 5erum and pr0te1n-f1ee med1a are pr0pr1etary, h1nt5 u5efu1 are 91ven 0n
the 1ac1t1rc cu1ture med1a c0mp0nent5 re4u1red, and exten51ve cr055-referenc-
9n1 t0 re1ated 5tud1e5 51 91ven.
Pr06a61y the m05t t1uc1ff1d 50urce 0f 11ec 1nfect10n t0 6e m0n1t0red and
c0ntr011ed 51 1ar1v 1nfect10n, and mamma11an 11ec 5en11 d1ffer n1 the1r 5u5cept1-
yt1116 t0 each v1ru5.7he5e d1fference5 are h19h119hted n1 Chapter 3, and a55ay5
t0 detect v1ru5e5, 6a5ed 0n PCR, 1mmun0f1u0re5cence, 0r cr055-c0ntam1na-
t10n 0f0ther ,511ec are de5cr16ed. 7he re9u1at0ry env1r0nment 9n1110rtn0c 1ar1v
5afety 5eu551 51 051a addre55ed 6y the auth0r.
n1 Part 11 the m01ecu1ar meth0d5 re4u1red f0r 9ene tran5fect10n, 11ec -m1
,n01ta211atr0m and 11ec fu510n are de5cr16ed, t09ether w1th the m01ecu1ar tech-
5eu41n u5ed t0 character12e the 5(cid:127)11ec chr0m050me5 and ,e9aen11 5a 11ew 5a the
preparat10n 0f 11ec 6ank5. When creat1n9 new rec0m61nant 11ec ,5en11 t1 51 0ften
nece55ary t0 1ncrea5e the 1eve1 0f 9ene n01tac1f11pma t0 en5ure max1mum pr0-
pr0duct10n. te1n 7h15 eu551 51 addre55ed n1 Chapter 4, wh1ch 5en11tu0 an a1terna-
ev1t meth0d t0 the c0mm0n n01tac1f11pma 6y marker n01tce1e5 and dru9 treatment,
1.e., n01tac1f11pma n1 v1tr0 6y c0n5truct1n9 mu1t19ene c0ncatenamer5. Creat1n9
y1h91h 9ene-amp11f1ed 11ec 5en11 5e51ar 5eu551 0f 9enet1c ,yt1116at5 n1 60th the
pre5ence 0r a65ence 0f n01tce1e5 a9ent5. Cyt09enet1c techn14ue5 that can 6e u5ed
t0 trace the yt1116at5 and 1nte9rat10n 0frec0m61nant 9ene5 n1 11ec ,5en11 5uch 5a
F1u0re5cence n1 51tu n01ta21d1r6yH (F15H), are de5cr16ed n1 Chapter 5.
8e51de5 rec0m61nant pr0te1n pr0duct10n, there 51 a need t0 create new
mamma11an ,5en1111ec f0r 60th dru9 and te5t1n9 t0x1c0109y and 5a 9ene vect0r5.
F0r the5e 5e1dut5 t1 51 1mp0rtant t0 create de211atr0mm1 11ec 5en11 w1th det1m11nu
9r0wth p0tent1a1, e11hw pre5erv1n9 0ther phen0typ1c character15t1c5 0f the .511ec
Chapter 6 de5cr16e5 the u5e 0fan 0nc09ene (5V40 7 1ar9e ant19en) t0 -1atr0mm1
e21 511ec and 051a c0mpare5 d1fferent meth0d5 0f d1m5a1p tran5fect10n.
Many 0fthe pr0te1n5 u5ed f0r re5earch, ,5c1t50n9a1d antdh erapy are m0n0-
1an01c ant160d1e5, and the re1evant pr0t0c015 are de5cr16ed n1 Chapter 7. Cru-
1a1c 5ta9e5 n1 ant19en preparat10n, 1mmun12at10n, 11ec fu510n, and 5e1ect10n are
h19h119hted, a10n9 w1th en2yme-11nked 1mmun050r6ant a55ay5 (EL15A) f0r
the eva1uat10n 0f 11ec pr0duct1v1ty. 7he 5e1ect10n 0f 1aud1v1dn1 c10ne5 6y -m11
9n1t1 ,n01tu11d 9n1ta1p n1 50ft a9ar, and u51n9 feeder 511ec 51 051a c0vered, 5a 51
the detect10n and n01tan1m11e 0f myc0p1a5ma c0ntam1nat10n and meth0d5 f0r
5t1 .n01tan1m11e
Prec15e, we11-d0cumented 11ec 6ank1n9 and 11ec character12at10n pr0ce-
dure5 are n0t 0n1y re4u1red 6y re9u1at0ry auth0r1t1e5 9n1110rtn0c the 9n15nec11
0f 610pharmaceut1ca15, 6ut are 051a cruc1a1 f0r the 5ucce55fu1 ma1ntenance 0f
11ec 5t0ck5. Chapter de5cr16e5 8 the key meth0d01091e5 f0r 11ec cry0pre5ervat10n,
rec0very, and yt111ret5 te5t1n9, and 5en11tu0 meth0d5 f0r 9enet1c te5t1n9 -116at5
Preface 11v
yt1 that c0mp1ement th05e de5cr16ed n1 Chapter5 5 and 9. 5pec1f1c pr0t0c015
f0r DNA f1n9erPr1nt1n9 0f 1am1na 511ec are c0vered n1 Chapter 8, 9n1du1cn1 a
c0mpar150n 0fthe u5e 5uc011t1umf0 and 5uc01-e19n15 DNAp r06e5.7he5e meth-
0d5 are u5ed t0 c0nf1rm the 1dent1ty 0f 11ec 5en11 (51nce cu1ture5 can 6ec0me
cr055-c0ntam1nated n1 a mu1t1purP05e ,)yt111caf and 051a t0 track en11 11ec 5ta-
yt1116 dur1n9 11ec adaptat10n, 5e1ect10n, and 10n9-term cu1ture.
n1 Part ,111 pr0t0c015 deta11ed are pr0v1ded f0r the 5tudy 0f 11ec 9r0wth,
,yt1116a1v meta60115m, and pr0duct1v1ty. 7he t5r1f 5tep 51 t0 determ1ne the 11ec
num6er and ,yt1116a1v and 60th d1rect 11ec c0unt1n9 meth0d5 and 1nd1rect pa-
rameter5 (5uch 5a 91uc05e uptake) are c0vered n1 Chapter .01 D1fferent 11ec
yt1116a1v parameter5, 5uch 5a dye re1ea5e exc1u510n, 0f 1actate dehydr09ena5e,
ra1u11ecartn1 ener9y char9e, and pr0te1n rate5, 5ynthe515 are 051a c0mpared n1
51ht chapter. F10w cyt0metry ha5 6ec0me a very u5efu1 meth0d f0r 9n155e55a
11ec 9r0wth and pr0te1n pr0duct1v1ty, and Chapter 1 1 5en11tu0 the re1evant pr0-
t0c015 f0r 11ec cyc1e ,515y1ana perf0rmance pred1ct10n, 11ec ,yt1116a1v and 5ta-
yt1116 mea5urement5, 5a 11ew 5a f0r 9n155e55a the 196 c0ntent 0f rec0m61nant
.511ec Dur1n9 cu1ture, 511ec can e1d 6y e1ther necr0515 0r ap0pt0515, the 1atter
6e1n9 a 4u1ck and pr0ce55 c0ntr011ed that can 11atruc 11ec pr0duct1v1ty. Chapter
21 de5cr16e5 h15t0chem1ca1, f10w cyt0metr1c, and DNA 9n11e6a1 pr0t0c015 f0r
eva1uat1n9 the rate5 0f 11ec death 6y 60th ap0pt0515 and necr0515. NMR 5pec-
tr05c0py ha5 the un14ue yt1116a y15u0enat1um15 t0 9enerate exten51ve 1nf0rma-
t10n 0n w1de a ran9e 0f 1mp0rtant meta6011te5, 5uch 5a nuc1e0t1de--ph05phate5,
NAD, and creat1ne ph05phate. Pr0t0c015 f0r mea5ur1n9 the5e 11ec ener9et1c5
parameter5 n1 perfu510n 610react0r5 are de5cr16ed n1 Chapter .31
n1 Part ,V1 the m0re t511a1cep5 (e1ther techn14ue5 n1 cu1ture meth0d5 0fce11 0r
n1 9n1tav1t1uc t511a1cep5 11ec )5en11 are .de61rc5ed Chapter 41 5en11tu0 the c0n5truc-
n01t and ma1ntenance 0f a n015ufrep 610react0r, 9n15u a de21d1u1f 6ed 9n1n1atn0c
511ec anch0red t0 m1cr05phere5.7here 51 a 11am5 9r0up 0fpr0te1n5 that are anch0red
t0 the 5(cid:127)11ec am5a1p mem6rane a1v 1yd1tahp50hp1y50cy19 10t150n1 (6P1). 7h15 en-
5e16a the pr0te1n t0 6e harve5ted y115ae 6y c1eava9e 0f the anch0r 9n15u -110hp50hp
C pa5e fr0m 511ec 9r0wn n1 n015nep5u5 0n 0r 5ed15e8.5erehp50rc1m y11arutan 9n1rrucc0
anch0red ,5n1et0rp the 6P1 pre-anch0r 5e4uence can 6e en91neered 0tn1 rec0m61-
nant pr0duct5 6y creat1n9 n015uf ,5n1et0rp thu5 9n1tat111caf the 9n1t5evrah 0fpr0te1n5
that are n0t y11amr0n (Chapter5 5ecreted 51 and 16).
1ncrea51n91y, human hemat0p01et1c 511ec are 6e1n9 9r0wn n1 v1tr0 f0r
11ec tran5p1antat10n and 5a 9ene therapy vect0r5. M0d1f1cat10n5 t0 the 6a51c
11ec cu1ture, character12at10n, and 5e1ect10n and expan510n techn14ue5 are re-
4u1red, 51nce the 1a1t1n1 cu1ture5 c0nta1n a heter09ene0u5 p0pu1at10n 0f d1ffer-
ent 11ec 11nea9e5.7he5e de211a1cep5 techn14ue5, wh1ch pr0t0c015 1nc1ude f0r
retr0v1ra1-med1ated tran59ene 1nfect10n 0f6100d ,511ec are de5cr16ed n1 Chap-
..°
vm Preface
ter5 71 and 18. A ra11m15 pr061em 0f cu1tur1n9 d1ver5e 11ec type5 0ccur5 n1
cyt0t0x1c1ty ce11-6a5ed te5t1n9. 7he re1evant a55ay5 u5ed t0 m0n1t0r t0x1c re-
5p0n5e5, wh1ch 1nc1ude dye re1ea5e, dye uptake, re1ea5e, rad10150t0pe and me-
et1106at a55ay5, are the 5u6ject 0f Chapter .91
n1 PaVr,t the effect5 11ecf0 cu1ture 5n01t1dn0c 0n the 1an01ta15nartt50p m0d1-
n01tac1f 0f pr0te1n5 are de5cr16ed. Many re4u1re pr0te1n5 exten51ve -a15nartt50p
t10na1 m0d1f1cat10n t0 ach1eve 11uf 1ac1901016 act1v1ty. 1ndeed, authent1c
1an01ta15nartt50p mach1nery 51 0ften c1ted 5a the rea50n ma1n why many 5n1et0rp
u5ed y11ac1tuepareht are manufactured 9n15u mamma11an, 5a 0pp05ed t0 m1cr0-
,1a16 .511ec Meth0d5 u5ed t0 detect the extent 0fpr0te1n 9n1d10f and the 5c1ten1k 0f
pr0te1n 5ecret10n are de5cr16ed n1 Chapter 20. 7he5e meth0d5 ev10vn1 -05101dar
t0pe5 u5ed n1 re91me, pu15e-cha5e a dew0110f 6y n01tu105er 0n acry1am1de 51e9 and
aut0rad109raphy. 515y10et0rP 0fthe dur1n9 pr0te1n expre55ed 51515ehtny5016 a c0m-
m0n phen0men0n, e1ther n1 the cu1ture med1a f1e5t1 0r dur1n9 d0wn5tream pr0-
.9n155ec n1 50me ca5e5 the act10n 0f 5uch c1f1cep5 pr0tea5e5 5a pr0h0rm0ne
c0nverta5e 51 a et151u4ererp t0 9n1ve1hca 11uf 1ac1901016 .yt1v1tca M0re c0mm0n1y,
h0wever, 515y10et0rp 51 an unwanted and de9radat1ve 5tep that can 6e de21m1n1m
6y accurate m0n1t0r1n9 and the u5e 0f pr0tea5e .5r0t161hn1 Pr0t0c015 f0r man1pu-
9n1ta1 the5e pr0tea5e-re1ated event5 are de5cr16ed n1 Chapter 21.
61yc05y1at10n 51 the m05t exten51ve and var1a61e 1an01ta15nartt50p m0d1-
f1cat10n made t0 pr0te1n5 expre55ed n1 mamma11an .511ec t1 0ccur5 n1 m05t
5ecreted and mem6rane-60und mamma11an pr0te1n5. Deta11ed pr0t0c015 f0r
pr0te1n 91yc0f0rm 515y1ana u51n9 yra111pac e1ectr0ph0re515 and ma55 5pectr0m-
etry are de5cr16ed n1 Chapter 23.1n add1t10n t0 91ycan var1at10n5, 6105ynthet1c
heter09ene1ty can ar15e fr0m 5e5ad150cy19 re1ea5ed fr0m 511ec dur1n9 cu1ture.
7he mea5urement 0f e5ad150cy19 f1u0re5cent u51n9 act1v1ty 5u65trate5, 5t1 ef-
fect5 0n 91ycan heter09ene1ty, and mea5ure5 u5ed t0 e21m1n1m 91yc051da5e-
dependent de9radat10n are de5cr16ed n1 Chapter 23.
n1 5ummary, 51ht v01ume c0n5t1tute5 a c0mprehen51ve manua1 0f tech-
n14ue5 that are 1a1tne55e f0r the 5ett1n9 up 0f a 11ec cu1ture 1a60rat0ry, the
r0ut1ne ma1ntenance 0f 11ec ,5en11 and the n01ta21m1tp0 0f 1ac1t1rc parameter5
f0r 11ec cu1ture. 1nev1ta61y, 50me 5n01551m0 111w 0ccur n1 the text, 6ut the au-
th0r5 have 50u9ht t0 av01d n01tac11pud 6y exten51ve cr055-referenc1tn09 chap-
ter5 n1 0ther 0fth15 v01ume5 5e1re5 and e15ewhere. We h0pe the v01ume pr0v1de5
a u5efu1 c0mpend1um 0f techn14ue5 f0r 5t51tne1c5 w0rk1n9 n1 1a1rt5udn1 and
re5earch 1a60rat0r1e5 that u5e mamma11an 511ec f0r 610techn0109y purp05e5.
7he ed1t0r 51 9ratefu1 f0r the 5upp0rt 0f 11a the c0ntr16ut0r5 and the 5reh5116up
wh0 have made 51ht v01ume .e161550p
1e91N 5n1kneJ
C0ntent5
Preface .............................................................................................................. v
C0ntr16ut0r5 ................................................................................................... 111x
7RAP .1 6N1L8AN5 5E160L0NHcE7
1 5ett1n9 Up a New Ce11Cu1ture La60rat0ry
e11ehc1M F. 5c0tt, Cather1ne M. 8ent1ey,
and Car01ynne .7 Mar5ha11 ............................................................... 3
Adaptat10n 0f Mamma11an 511eC t0 6r0wth n1 5erum-Free Med1a
Mart1n 5. 51nac0re, Den15 Drapeau, and 5. R. Adam50n ................ 11
1ar1V Eva1uat10n 0f 1am1nA 11eC L1ne5 U5ed n1 810techn0109Y
A1a5da1r J. 5hepherd and Kenneth 7. 5m1th .................................... 23
7RAP .11 RALucEL0M 5D0H7EM
4 0pt1m121n9 6ene Expre5510n n1 Mamma11an 511eC
Luc1a M0nac0 ....................................................................................... 39
5 Cyt09enet1c Character12at10n 0f Rec0m61nant 511eC
na1r01F M. Wurm and 01etmar 5ch1ffmann ....................................... 49
6 n01ta211atr0mm1 5trate91e5 f0r Mamma11an 511eC
J0hn 5tephen McLean ........................................................................ 61
7 M0u5e M0n0c10na1 Ant160d1e5: n01ta21numm1 and
Pr0duct10n Pr0t0c015
Mar9aret 600daf1 ................................................................................. 73
8 11eC 8ank Preparat10n and Character12at10n
5tephen J. Fr0ud ................................................................................. 99
9 DNA F1n9erpr1nt1n9 and Character12at10n 0f 1am1nA 511eC
61yn N. 5tacey .................................................................................... 117
7RAP .111 CELL N017AuLAvE PR070c0L5
10 11eC C0unt1n9 and yt1116a1V Mea5urement5
M1chae1 8ut1er .................................................................................... 131
/x
x C0ntent5
11 9n1r0t1n0M 1am1nA 11eC 6r0wth and yt1v1tcud0rP
6y w01F Cyt0metry
M0hamed A1-Ru6ea1 ......................................................................... 145
21 Mea5urement 0f 11eC Death n1 erut1uC
Af5h1n 5ama11 and 7h0ma5 6. C0tter .............................................. 155
31 Nuc1ear c1ten9aM Re50nance Meth0d5
0f 9n1r0t1n0M 11eC m51106ateM
Mar1a L. Anth0ny, 5hane N. 0. ,5ma1111W
and Kev1n M. 8r1nd1e ..................................................................... 165
7RAP .V1 751LA1CEP5 5EUQ1NHCE7
14 9n1rut1uC 511eC1am1nA n1 de21d1u1F 8ed React0r5
Annette Wau9h ................................................................................. 179
51 6P1-Anch0red n015uF 5n1et0rP
L. Ma1c01m Kennard, 6re90rY A. ,ee21L
and derf11W A. Jeffer1e5 ................................................................ 187
61 6P1-Anch0red Harve5t1n9 5n1et0rP fr0m CH0 511eC
L. Ma1c01m Kennard and Jame5 M. P1ret ...................................... 201
71 c1te10p0tameH 511eC f0r ra1u11eC and 6ene 7herapy:
.1 8a51c A55ay 7echn14ue5
519ma 5. M05tafa, D1ane L. Hevehan, 70dd A. McAdam5,
E. 7errY Pap0ut5ak15, and ma1111W M. M111er .......................... 211
81 c1te10p0tameH 511eC f0r ra1u11eC and 6ene 7herapy:
.11 Expan510n Pr0t0c015
70dd A. McAdam5, E. 7errY Pap0ut5ak15,
and ma1111W M. M111er .................................................................. 229
91 yt1c1x0t0tyC 9n1t5e7 9n15U 11eC 5en1L
L0rra1ne D. 8uck6errY .................................................................... 239
7RAP .V 7CUD0RP N017AULAVE 5L0C070RP
20 9n1ru5aeM the 9n1d10F 5c1manyD 0f tnan16m0ceR 5n1et0rP
5ecreted fr0m na11ammaM 511eC
Neff J. 8u11e1d and Adr1an R. Wa1m51ey ........................................ 255
12 10rtn0C 0f 515y10et0rP n1 11eC :erut1uC
U5e 0f 5r0t161hn1 and En91neered 11eC L1ne5
Mar1a 6. Ca5tr0, 51m0n W1ndeatt, and Marce10 J. Per0ne ......... 263