Transfection of Human Lymphoblastoid Cells with Herpes Simplex Viral DNA (Epstein-Barr Virus/B Lymphocytes/Mitogens/Infectious Centers) GEORGE MILLER*T, P

Home , Transfection

Proc. Nati. Acad. Sci. USA Vol. 76, No. 2, pp. 949-953, February 1979 Medical Sciences Transfection of human lymphoblastoid cells with herpes simplex viral DNA (Epstein-Barr virus/B lymphocytes/mitogens/infectious centers) GEORGE MILLER*t, P. WERTHEIMt, G. WILSON*, J. ROBINSON*, J. L. M. C. GEELENt, J. VAN DER NOORDAAt, AND A. J. VAN DER EB§ *Laboratorium voor de Gezondheidsleer, University of Amsterdam, The Netherlands; §Laboratory for Physiological Chemistry, University of Leiden, The Netherlands; *Department of Pediatrics and Epidemiology, Yale University School of Medicine; and tHoward Hughes Medical Institute Laboratory at Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Dorothy M. Horstmann, November 14,1978

ABSTRACT The "calcium/dimethyl sulfoxide shock" the pellets and centrifuged for 3 hr at 18,000 rpm at 4°C in the method of transfection was adapted for use in human lymphoid Beckman 19 rotor. The pellets were resuspended in 1/200th cell cultures. One microgram of herpes simplex virus type 1 DNA regularly initiated virus replication in four lymphoblastoid cell vol of 0.15 M NaCI/0.05 M Tris, pH 7.4 (TS buffer) containing lines. Per 105 cells exposed to 1 pg of DNA, 0.5-5 cells formed 0.25 M sucrose and placed on top of a continuous 10-50% an infectious center. The minimal infective dose of DNA was (wt/vol) sucrose gradient in TS buffer. The gradient was cen- approximately 500 ng. trifuged in the SW 27 rotor for 1.5 hr at 23,000 rpm at 4°C. The virus band was harvested and dialyzed overnight against TS A reliable method for transfection of human lymphocytes by buffer. DNA could have many applications in the study of lympho- The purified virus was concentrated by pelleting for 1.5 hr tropic viruses and also might be useful in experimental im- at 35,000 rpm in the SW 41 rotor. The pellet was resuspended munology and genetics. To initiate studies of this problem we in 0.05 M Tris/0.1 M NaCI/5 mM EDTA, pH 7.4 (TNE buffer) chose a system that would maximize the chances of demon- and viral DNA was released by addition of 1% sodium dodecyl strating expression of genetic information after introduction sulfate and 2 mg of proteinase K per ml. The DNA extraction of naked DNA into lymphoid cells. The DNA of herpes simplex mixture was held at 370C for 4 hr and then was gently pipetted virus (HSV) was selected because one assay for expression- on top of a 10-30% (wt/vol) sucrose gradient in TNE. The namely, production of complete progeny-is simple and rapid gradient was centrifuged for 3 hr at 35,000 rpm at 180C in the and because several methods are already available for detection SW 41 rotor and was fractionated by pumping 40% (wt/vol) of infectious herpes DNA on monolayer tissue culture cells sucrose in TNE into the bottom of the tube. The absorbance (1-3). The ability to assay HSV-1 DNA independently in per- across the gradient was monitored with a Uvicord III (LKB missive cells provided a check on the quality of the DNA and Instruments) and recorded on a W and W recorder. High mo- on the adequacy of the transfection technique. HSV DNA was lecular weight DNA was collected and dialyzed against 0.01 chosen also because it is a good model for future studies with M Tris/0.1 M NaCl/1 mM EDTA. The concentration of DNA Epstein-Barr virus (EBV) DNA, a molecule of the same size. was determined by absorbance at 260 nm. The yield of high molecular weight DNA was 6-34 ,ug per batch. The DNA used MATERIALS AND METHODS for transfection experiments was stored in aliquots at Virus Strain, Propagation, and Preparation of Viral DNA. -200C. The Lovering strain of HSV-1, originally from a fetal enceph- Lymphoid Cells. Three lymphoblastoid lines, designated alitis case, was passed at low multiplicity three times in the X25, were derived after transformation of lymphocytes from AH-1 and once in the BSC-1 line of African green monkey one umbilical cord with a passage of the B95-8 strain of EBV kidney cells (4). The virus was plaque-purified in BSC-1 cells (unpublished data); the fourth line (X50-7) originated from under 2% methylcellulose. Two batches of viral DNA were another umbilical cord. These lines are not spontaneous pro- used: batch 4, prepared from virions propagated in BSC-1 cells; ducers of extracellular EBV but transforming virus can be re- batch 6, derived from a subclone obtained in transfection of covered from the lines by the technique of x-irradiation and BSC-1 cells, was prepared from virions propagated in BHK-21 cocultivation. All the cells have EBV nuclear antigen, and re- cells. ceptors for erythrocytes coated with antibody and complement Viral DNA was obtained from virions by the procedure de- are found on 90% of the cells in each of the lines. Some of the scribed by Geelen et al. (5) for preparation of infectious cyto- cells in the X25-9 line adhere to plastic surfaces, but adherent megalovirus DNA. The starting material for each batch of DNA cells are not found in the X25-4, X25-6, or X50-7 lines. Raji and was 6-20 roller bottles of BSC-1 cells. The medium was re- BJAB are Burkitt lymphoma lines; Raji has the EBV genome moved from confluent cultures, which were inoculated with and BJAB does not (6). Mixed mononuclear leukocytes from about 2 plaque-forming units (PFU) per cell. After 1 hr the umbilical cord blood were prepared for culture after isolation inoculum was removed and 50 ml of Eagle's medium with 2% on a Ficoll/Hypaque gradient (7). The cell lines were main- calf serum was added to each bottle. After 40 hr, when there tained in medium RPMI 1640 plus 10% fetal calf serum and was extensive cytopathic effect, the culture medium was de- antibiotics. canted and centrifuged at 4000 rpm at 4°C for 10 min in the Virus Assays. HSV plaque titrations were performed in Sorvall GSA rotor. The supernatant fluids were separated from BSC-1 cells under 0.9% agarose. Three days after infection, neutral red was added at a final concentration of 1:10,000 to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: HSV, herpes simplex virus; PFU, plaque-forming units; vertisement" in accordance with 18 U. S. C. §1734 solely to indicate EBV, Epstein-Barr virus; EE-10, Eagle's medium with Earle's salts this fact. and 10% fetal calf serum; Me2SO, dimethyl sulfoxide. 949 Downloaded by guest on October 1, 2021 950 Medical Sciences: Miller et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Sensitivity of various lymphoid cell preparations to infection with HSV type 1 Infected centers, no./104 cells* Type of cell Treatment or cell line Exp. 1 Exp. 2 Primary mixed human umbilical cord mononuclear leukocytes None 12 18 Agingt 170 161 Mitogent 373 304 Mitogen + aging 445 ND EBV-transformed human umbilical cord leukocytes X25-4 400 1250 (lymphoblastoid cell lines) X25-6 495 726 X25-9 430 ND Burkitt lymphoma lines Raji-EBV genome-positive 3 13 BJAB-EBV genome-negative 8 23 * At 24 hr after infection with t2 PFU per cell, infected cells were incubated at 370C in antibody for 1 hr, washed, and resuspended in medium. The cells were counted and serial dilutions were plated on VERO cells. A methylcellulose overlay was then added. After 3 days the overlay was removed and the cell sheet was fixed in methanol and stained with Giemsa. The wash fluid after antibody treatment was also plated and the number of plaques that developed was subtracted from the number of infected centers. ND, not done. t Cells had been in culture for 5 days before they were exposed to virus. t Cells were treated with staphylococcal phage lysate (150 jg/ml) and 2 days later were infected with HSV-1.

a second agarose overlay. Plaques were counted between days and the precipitate were incubated for 3.5-4 hr at 37°C. The 4 and 7 after infection. HSV-infected centers in lymphocytes suspended cells were removed from the petri dish, and washed were measured on VERO or BSC-1 indicator cells under 1% once in EE-10; 25% dimethyl sulfoxide (Me2SO) in Hepes- methylcellulose. Plaques were fixed with methanol and stained buffered saline was added to the resuspended cells. After 4 min with Giemsa after 3 or 4 days (8). at room temperature, 3 vol of Hepes-buffered saline was added Transfection. A precipitate was formed by adding viral to dilute the Me2SO and immediately thereafter the cells were DNA, 5 .ug of calf thymus DNA, and 125 mM CaCl2 to a final washed once with Hepes-buffered saline and once with EE-10. volume of 0.5 ml in Hepes-buffered saline at pH 7.05. This They were resuspended in EE-10. X25-9 cells that remained reaction mixture was mixed gently and allowed to stand at room attached to the dish were also washed, treated with Me2SO, and temperature for 0.5 hr. The infectivity of the DNA was assayed washed again. The cells that had been in suspension were then on freshly trypsinized BSC-1 cells in 60-mm petri dishes. returned to the original dish with its adherent population. To transfect lymphocytes with DNA, the cells growing in suspension were removed from RPMI growth medium and washed twice in Eagle's medium with Earle's salts and 10% fetal RESULTS calf serum (EE-10). The cells were resuspended to a concen- Susceptibility of Lymphoid Cells to Infection with HSV. tration of 105-106 cells per ml and 5 ml was placed in a 60-mm Different types of lymphoid cells varied considerably in their tissue culture petri dish (Falcon). The preformed precipitate sensitivity to infection with HSV. All were less sensitive than was added in droplets to various regions of the dish (9). The cells monolayer tissue culture cells. Some representative data are shown in Table 1. The number of infected centers was measured 24 hr after addition of 1-2 PFU per cell. Primary lym- A phocytes were relatively refractory to infection. However, if the primary cells were "aged" in vitro for a week they became 10- to 20-fold more susceptible. An increase in susceptibility to infection of similar or slightly greater magnitude was ob- served in cord leukocytes treated with staphylococcal phage lysate (Delmont Laboratories, Swarthmore, PA). This lysate is a mitogen of T and B lymphocytes and null cells (10). Lym- phoblastoid lines resulting from in vitro transformation of umbilical cord cells by EBV appeared to be more susceptible to HSV than were primary cells from the same origin. Lines derived from Burkitt lymphoma were relatively refractory to production of complete virus (11). Thus, lymphoblastoid lines appeared most suitable for initial attempts at transfection with DNA. Characteristics of Infectious HSV DNA. Representative profiles of two sucrose gradients on which HSV DNA was pu- Top Bottom Top Bottom rified are shown in Fig. 1. The DNA preparations used for FIG. 1. Velocity sedimentation of DNA extracted from banded lymphocyte transfection studies were full-length genomes HS virions, across a 10-30% sucrose gradient. Absorbance at 254 nm harvested from peak 2. The mean contour length of DNA was measured with a continuous flow spectrophotometer. (A) DNA molecules in peak 2 was 15.9 times the length of the PM2 prepared from virus harvested from eight roller bottles (batch 3); there marker. This corresponds to a size of 101.36 X 106daltons. DNA was about 6 jg of DNA in peak 2 and about 3 jg in peak 1. (B) DNA prepared from virus harvested from 20 roller bottles (batch 4); ap- in peak 1 was 4.15 times the size of the PM2 marker and thus proximately 13 ,ug of DNA was present in peak 2 and 7 jg in peak the molecules are approximately one-quarter full genome size. 1. Most of the full-length HSV DNA molecules found in peak 2 Downloaded by guest on October 1, 2021 Medical Sciences: Miller et al. Proc. Natl. Acad. Sci. USA 76 (1979) 951

- - -.r " - '. I .- ,. ... I . - .,- . -- ". %..I,1 ;.7 --"'. -V, :. .;, .. .'. t. i.. II.10.". '. -.' ". % '.., -.- ": ...". -0 "". ,, ,- V- -._-,-, .,. ..., 't'.. .. Z V il ., ''-,,% ,; %.;-., 1'..." .,.,, "j.. ' ,'..1-..," ,...., .. ,--' .--'-- : 4.. ..,, -:,,,,--,'I..ji " N :.,..1- ,,,,,,T-4.,..!-o'-.- ;' ";...... " ... :7 .-4; ,!.. " -,.....4i. .! .... '. .'_ I,.- .,. .'-." -- ,,N, ,...-,..wk. ."-.:., 'I . I. ,.C.11-..-_-...... ; ,.'..-.,.-i., -, :,4:..-.----. -- - .;.W, --,,11.-, 1, .I..,-.... ii ",-..--.7-. 1. .,,,,...",.....,4...t,.. .!'- -. ", -'. . ,-,Ii.";,- ...,4..,.rc , . w:,.,.. "., ...-,, -..,I- ., -.,-.'5:.-- -7. J ..-F -4 ".-.-Z. .';. 411'...-..-L.v ..--I-I...-:-t-..- . ..-...,-I" ,, .-I",..'-.:,, ,- .,;.,,, ,.j ,,,-A, i.-A..'.. '_ '4; " ` ". 4, ...I'- .4 '.,Ik ." ._ft6,; #iL.-"---,7,--.1-- - I- ,- ,-#,C1;."' 1..:.,....,+ .I.,., ,4.,*-,.- ,---. '-.' , , f.,; .... .__ " -.-'% 1% W,." -,.., . ._.,..--,.'i-.--,,,I ,.:...... ,-.." 11. .,,.'7 ".....il",L' ,4,.- ....st. i--,- " ..,.,.,."t --" "-ri ;"".,,. :i.-;--.-,-....;,., .,., .k. .. "-.1 " ..: 'r '.. ..'.- '. ,..,,-.' -;., ..0, -.!,., !,--% A I.' .., .'. A-.%:,,.,-. %I I.-....Z.-. ..I"..--..-II.,:.a. ,. ..,-.:.,L 4%;--,".'.W,'. 'A...,,; 1i,-, . ". ".. .- .,s--:i..;.-.. ii* ...... -, ;,. t- ,..?, ..- ., -.r '.-...... ! 1. .. .-,..., ....,... -,;,.., 7 -.,'.i. .,_,- '. ..- ;.!,__"..,.. ,,,-.;, ,"-., .. '... ", . .,,,.., r '. A.; ...,.,, _;. 1, ,,I.. .'. .,-.I.? -,-.41O" .:. .,% -a .4 '.. -j.4. .j,...-1,..-, "..,.. 1. ., -'. ".,I...-.4 ,....It ia,;.-,,,*-1,.... .1, -,..-.,e,....- ,.-!,..4-*-I',-,---,,,,,. , ,..,. I .!-, . ,; ;..te -..--I- ..v .1 -,..I. -." -.".!.:- f-"'4t- "I I-o...." ,;;.. 't. -..q_ . ,'. . - ."; ,.... 4s.,._S.t:'11.t, ". 1. ,-.. `. ;,:., ,%, "I It.-,,-I.'---.','.-- ...-,,-....,?"...,,iK-.': ... "t "',... ".,.--.,-.---;-,r..; id-';.- ... .i;, ".- It ., :. .," '. I..- t, ... .,:,-._'",...,V..-,,..,.",.I-.41 .-:."I".. ,-, --..'.-.; -.* .."',.;:, -., ;.. . .,. ;.,.: ." ... 1:11 .r .. ..: -".,,.,, ...... -'.'k- _. ,!,.. ,,.;.., -,.. ..,. .1 ." '.1.... ., .t.,_i':' ., .,--ir % ".,.14 _., -.., ...,,!-'.,ItV.,...:..,. .. ,.,- .. .1. .. :,.,.,-, .. -....'-, ,., ." ,-,,.,..,...v ,.1 .. ,:-,. . ..; .. ... , ,..,,..,,14.t. '...-" -. - .. ., ",),I .. .,--.". .. t., '--4. . .. " -. -,,,. -- ,""'4.- -t, .let..-. -4, '.,I.."I.i. ..--,-.. e .!. ,. .. i"'-,. .,:.. lt., '.. " --z'.,-,:.I..,---I..'1. ,. "' " %:.I...,.: ,- -!,. '. .I -..,.#...&-.Ie-`-%.-.-,-,... - ."..WV..".-.-,;,...,I--'.".,;,., 1.-- ..; ,.,-,..,. !-S,.-.- . I..-,,.. :. -,--. ., -.;. -_---- ....-..-,. .,-.... .-- --IIV,- ;,.-,,.,.---,.;..,,...2 -,. ,- . ..;, ...-.II..: -. ,,I.:.-, .- .... ".:..---...,,,:.., -.0.;, .-I...r-L ,.N... Tr -.-! . ", .., ".. - `-, W. ._'._ .% .., ..,--'1.-..1'-.I..,Z...',, .1 .-.. :. '.i.... .1. _.....--..., .: "...... ,,-.-.1".-...... 't,-. .", I .,.'.-'...... -1, , " .,., .,'--,.-,i.- -,-..-.. ___..-.,-`,'.-- .:"..-. ,:,.,11, - -,-5 - --,:.F'.. :-.,.,.!...... --,I. -.....,.... I.-. , ;- 4P ,:,..;, ? t...,*,...."-.._ *,%-.., .-,- -. .I-&.. I.,.. ,.,v. .. -,;, j %. "t ,0'-..-I;,,. ..,vi'-- -'..ft '.x "., .1-'. -. ,I".;...,..411.I...:,..-.. -- -' '. ,.-.,-,qf....'_.,.F.I -'C. ., ...-,..I...,--ic-" -.. w 1:. .... ;7 ",..,..., I,.,- I..'r &.!" -... .I---..X., - 1, P "'...... ,;e .. ., ... "..., v 4 .,. -';. -. ., .. -. ."".. .-,' " 4, - .,,_ ...... i`-.,:,. .-,4,,.,''''i.nl ,. .j ..I..." . ".. %, -,- '. "r -,",. "I'..'.. ,-. I.- -,...& -, "s -i , .:, 1..__4""!.:; I-, _..%; .,-.. ", .,--;....- I....ir.z.,!.,!....,.,;-$,,,.,.I,- .,.,.!.. ,!- -...... '..jr ...... -....",.I,:.,.. -.,I-..,,,,'-,.. ...- _.,I.1'.I. ...,: .,.,,...... -1. '. .,,, ... - -..,.. -',' sv.*- .,. .., e, -,--.,',.. . ., - --.-.-,:.',',.--.-..-. -."I-,...", ,.!.. ,- ....,.-I..,. -I,,..---:11. 'I-117.. --' - .r., -V ,.,t-' ." :.1- .,..;r., I.,"i Z. Al .,'. a. ,. ,,. ;Z, - .,,..., ..%. .v ... .--..N..'....-,...:. -."'e, -".'- .,'.' I., .." ., .,.!." '. _."..'.1 ,!..t.. .,. I.-,.,;.. . -,_,,,, ...4,.-;,.,- r ;'...... ,,*;. 'O-I-,,,_...--- . .. .4.- ". 4'.... I"W'I.,..-..", 6. .... Z..W'A' %-.-' ,..-- '.! ..-....,I.-, -,;. ,- '. t .. 4. .,Yt-i ": "" ,-vi,-_ f..ftt.;, - .1.,..,., .-'!.' .-.;. 6.'...." -it. Ir: i .;,. -, '.' .. ...,lb."",Z."....f -Z .. `.- ".: ... .; ..,'. -... ,-' .. ." .,...4,. .4."*',..,,...'.I-`.. .:..; -- !:...,'....--.:%-'..,I_,,V!tj!.;...,.,. .1(, "_ , .-,_ -,4 .-i, -_ .; .. V ..4.-,.J'...": ._... -. W . 1, -,,. .- -'. ... .e .", .-.,.,. .... Zi,1, .. -,..,, 1. .-..---,-,., -,.i:...."'!.... "',,.-,.::..- ..,:. , -.'. .;.-.;, .,. %;..-.. ._...'.,.-,,"..I.P.., ..,,. a. -", i. - ,,...... "...." ,.. ..,. ...* ..-;2., .. . !k.. . -P ,,.%'. , "..ILI,.,k:. .. _.- ,.U. .4. ...1.'.-,'I, ;;..-..., V. *-44.116'w. ..--'c.'.,i_.,_. J ." .1i "- .,- 4.,1?.".i`.., "I",..., .- . .I..1 , -,! ii':. ...zi,4V "', V -..." 4 .. .;...... ,,I-. - o ... " '. .. 1. _;_,.., O., ,"".I ... ..!. ., .... ,.e '. P .. -..'! ,... ,..1-"---,!,;. .,,,..f.. " . .: ...... ,-:,W,,lb..,% '. ..,".,Al .0,.,,1,I,;,'. '.....,.-:";4;...'.-1,.-I... ..',-:;,..1...-..:.` ., ;_ ..'.. -1,,;I:Z ., - ,.>,.0,..,..I_--4 " . ., .' .,. ., . ..- -4, Z.-.:.,.-,.F, ,-, "'X--v.-I.--..'r..",-...,.I.,-.-.I.-,., 1, ,., ,i,.. ".". '. ,,. I, ", I'.-4 .-I...;..,.. -.%- .-...-,,P. " ., ... .Z-,... .., -I.- .I-,.,%t-%- ,.4 .. ..,'..%:,,-,-!..'7 r-., .. .,.%.,. -1. .L 111, -;"-.-,... ei-..- .. :. '4 %>-,-;!oL."IZi".:,., -,. !,.--,.;;..I;.. Iot-. .,'..., ,. .-S 1. ....'. ,.,I,., I,". .,?,,., .. -.-Z.;,- '-.I ?14 .: -,., .I... -,..,4.," -.;, -!"..%,.,,'16I,%. ...,...-..-t_-".. I.- 1...' -....,..1'7-.,.4..,.,1'. 11 ", .; '. 0. .-, - -,.., .- ',-. . .. t, .. .-;,. :..-. Ar...... ,. . .. ,.,... .'.-..IC-",-I,,vt%,..'. 1.,," .,,...,,.;, t. .:1..,-,....4'..".i,... --..,..-,.-. ".,.-'-,.,.,:, .. 1....,-"..' "-,-..- 'e. ,,,.-8..-..I.i, .:,..' '. .."-.eI... ..! --.. ,% . ". - '..'--),,,".91'".. ,.-_", .. v'%.' ,, --_4;!,.. " -. -...v".,;., '-,..vl "". ,$-";...:,.. -:Ile-It...... W..',,-,-" -.. .._. '.---I-.,.: ,. -II.;.;..", .,.-;-,...., _!., ,-...... t .. ;,, k... 0. -,' -- _% ., -,.L ,,..,-%i't-....._-_.-...... ,,;., ,c . ..A 11 A ..'' ...%...,.# ;'F, ,e.,-.",41.- C. ,L"., .. .,._-'';.'.,..,... .: .'.4.4... ,,t.%. -.....:.. .!. e1,..." -` ".,--,.--,-- .--..",!,:..-,-`...... 4.-.1''2'."- t'...-..: .. ., "I-..., 4- ."Z',.. .'-,..I,..,, r., .:..I- -.,-:, 4- .- ,* 4-,7::%-, ...,,..,,,. _ ,-.'.. .. 't,. ,'. ,1-.,.-.4-,C:,." -,j ., .II.,.,...,. ...,.,....-.1'.,...;*,."', ". L',.,,....-. ,it,; II.-...... ,;,.,v." ; .., .,.. .&.., ., -.__,; ..I --.. ,...... "", , , ;. i". %". -,.` t.'.- .. .:, ,.. '-I..4. .:1 .A- -,, .,--o-;. o" ,V 1;I,-., .S-.,it&_ ...*",,,..4..1:..:,..! .. sea, _.: ,;,..,.:.,..%. %-:;..1. i..-,,- IiA..I-, Al -i" ., -, -v.. $.. .. ", -t 1'. .j-.;-..:..?, '. _- %q..-.., .,,;., ;. --- -'j. ..V.i.-N--,-r... "----11--#-,,._-.-: --t..- .,. .... - ,., I -?..,Jt % ._., ...-. - .., '''!-,. -.,:. ".. .,- t, .-.,.'-,. ., -..:Zs,. 1.-%;Q.!-..-;I.i.,... .r, .. ,- ..,i-,C..-"Ir-0..'Jg-, .. -,.,V ,...-,.,,12.." -,;-',..-..,y -.-.,- .1, .:i.,.ir .,.,,,.. .>' .,"O.-.%I.... -1 I.."_; - -.?-...- .. V1. .. .",-.1.. ".,...%, . ,,. ., L,.,.,..,51 -'.-'%-'-,. 7,-.. I.,-:.,-% -...NV, .;.:,,.1 7._- .....W..-.,..'.V - " - ..: ..-"; .....".....-.." - .4 -1_. ...:.,.-.!.. _ -W ,7 .1 %, .- ,,,,.,,, ".., ... ,...,F,."--., ._'.".;, -, 41i. ."'11k.. 1, -1 .r .. _... 1 ..:-,.. -,., ... ,-. .: .-11-. '4!.., h_'.,.'_-- .. .. f. .e1..'....'-f... ,.,-, ..'; """ 't 1-.... --.,--... -...:.i1.: -I? -!,... .J I ,- I." ".--,-_..._,.,.,. .";.-1 ...,.,, f.l - 1.....".I-. '..-'. .'' -4-',""i.,V ,-,- .4-..w...'.'v.-...-, -s"', - -....,, ., - -:. ..I-,. ..A.;...-', -k 1z .,,,.-'... . ,,...-.'.-% .. 11. -:-.:,j ,,.'i_._.,:-tr-. -.., ,!stripe -.11-11 -:--_.-... '. '..P- ..,.,. -, .,r.,!. .., ,O - -:-:-.!,.IL...... ,. .1%, --."",,",,..,.I" % ,... '- .'..,?-1;I,...'ti., -4 * -, -., .P 4. -.? t.,,k-,_..v " It -....I`.,..-C !..,...--.!Z, & ;V ..,. ,I'... .,-..."L t.''....1 ...,r14.,Z ,L-:I-.i fz ..1...... - 1,; I'. ...,,-,,'. -- ..,.,. ...,,,:,.,..;.....1". -..1.-..%.,.,. `f.I.,:.:.r ...... -,. '! --r;-.41..t,.- 4q,-,.,...-Ile L. '. ,,,..._,.. .., '.. ..t. -!.;.,.. .-: ... .. 1.' ....,.17,. .;...I .ill. .". - '',...%-.., ";,. .-r..,..tl z, j. A ,4"".,I.q!.; -1 11, I ; ", 'I .%:lft.-.,.,--i .1, 11,...:..--,-M-.- ,.-'T..., 1.. ;,&. '. ...,:!..,_.C .- ,,L-...I..'L..-..'.J-.1'. ".,.I.. .;1.".6.I..-,;.-,, .t .._., ... -,. -,;,-., ..,- %".:;r 4 . ". ..-.t..P., .. ..-_, _-,".%.v.-.-.. . .J. . -.V,...,,,11.,,,.,-..'.,- - ."..-,' ., .,. " "- ,4-, e:... .1:--..-, .'t. -,.;,. ,..,Z - ...1.,S .N,_' ..-..,.I...-1.,.. .",i-;-.-- ,.:o.L.4'....--, - .'.. .,I'A'A,-.14..1 -, ".4..R...-;. ,...---i-..1 -`.',.. ,,--, .t ..,--i---11..%I._..,,..,..'. .,%. -;,4'. .:.- ."7o!-;"K..": .. . ..v ., .. .-,',,:..-i;,,.-,C--....." ....'; . -.1..410.,..,'....'t,.-,C I'll. I-.: ., ,". 1,I.ip."-In.-.-..,,..I," "'t., .:.,,.;-"....,-.. ,.--.1,,,.. .,I-., .5.- .-.. 1...1,,.-..L-&- .",...--,,,,-...`41., . .-,-"....I.- ., "..., .j., i... W,.4. .--`.N -'. ..:,.'..n. ..1.,....,,..,.-!, "..,e ;A..."I.,'.';. .. -1 -. .:,4 - .'-. .",.'.,,... -,;..1..*'.--_4._-_,..!.V7,---,; -0 4 -.4 ..1,-'..,94....I_-i-...... 91 -...,...... -..%,, L.,,,,I ---.'. -,..--,- .j .. ., .jr 'jV' 'L ..., .-O.: ....O-'. ..4.:;..I,''-.4,j'._..! ","..111 . .I._.",._ ...., -., ,,.,_....,-.,...-.I...... %, .... '_j, It-.,:.,,- ,;,., ."'..-,!,:7. .%.. ..:., -:,.....-,--%.- .: "',,,I.....;. 1!'. -..."%.. .. ."- .,- ,-.` * .._ ,.a .... - . -*&,... ,.,, ZL, .'. ...-.-,. ....--.;, ' ."- --.. ,;.: .,. ..".,-.,NI,.'.1.C_4"T.,-, ,.'. -., .,3., t.., ..., .. .k-...".., . "'. " ..%L ..k-.6j"". 4. ,.. .,,-,.,;!. 1...",.,'k,.!..%'.,.'11,.; -1 'A'tl._..-. ,.. ,.I..-'.'?" " V. _;; :5. 11,,I-.,& ,I ,-; 11-;.'._. I,-,,,Z;.i .'-,., %,-,. ,. ., -,.----%.. .; 'I....,._;s .__ ...... -,,'.-...,I;4..I.I --.'I.".;. .'I,.t.'- . .Z, 4, .7. .I.`.:- 'k- --. '. ..;. .A. ..,, '.., -, ,-,.- .", I,-,,'- ..n%.'! I .4 - ",".:,-',,.--- ..L...i I'.,-"--".,.'P.,..,.r.,.., 1. ,,. _',...i'. j'_ L" I,. ;.._ ,. ."...Z.....: .-,.-,-.r,.,.'.'L'.,,-.-'- ,"""'t.11.;.. .;,.LI 1...t,.,.. .,- ,.,--- -, ,- "L.-,..,.'.?v.j..11 .. -.- -V--11.Sxi-i...... ".'. .!..-,.. ki; ., ,P4!, W".f...... ,-..... %,, ...... -, ..-_."..'-j". ...i-'.,-...-O, ,:. ..,4. , ... -'.:'.,.,%,,.-L.,Ie.,..'-"., .. "I,'' ...." .v--'.-I' ':,.J' z...-,...N.. .., .,. L'...,.i. -.".., -,.,..- :7. ., - .-,, I .- .!. 4-,,::,.%:_*.'!.i,-Wo,%-,,..14,.-, ---"'."1:t..'..!,.,ti` :,: '. ,,,`F. t,,.'...-.:-. .,....;.- ..-:1.._k '01 ..1I.. .a,.,.I.. ,. 4.. ..,..---- ;,,. .;.I,". -:,%%V,.-.:.%,",':-%-.--...r -,i4...rf". ..,.-:.-. ..- .._ , %.- .. --'#",. .. .'_' .. . ..,,---o&, .,. "(.",#----,...... ".-.-;..:.-.-.4---_; ... i,-"..,;;.,,%,..4 .,L.--:;. ..1_,_;.,I,.-; _'._-,t. --.. .. ;,.! .4 : .-. .., ,.... ,:-, _t,?-..-...1:0... V ,5.;.,., "-I... .,. ". z.-..-. '. ;,, ... . .;-, -v,4....,..'k.; .7L .,. .,--,..,:.2..: -,..I,..',;,.0.1.: "'...;,._'k""J'.'..6,. '.'" "..,.-....It, ,.g. " "76, - :,. Xt '. ....I,%,_rt...... ,.4.,---, .".,;,.. .-,.-.. ,",.1.i...'.. '- L-1... ,.." .IZA ".--... ,,v I-,.,.... ., .:,... .. _ t, .r -. .-- "e.. .-.,i_,!.-,:., --.,. ', ...,;,!!.,Ii.f-- kZ ,--:_". ,,.-.. '-.,' I, .-. "P" .,..-., II..-",..,.,.;..I"f...;t-.4 ,.r..%,,."_.'.;. ..'..';,.L-..%,,,,% ". '..-.-.,,-.-,-.,.,- ,;...... ,Z...... -''o--, I. --N- 1_.-, , ,.,I.1 .' ,..;.., 4-M ".,,.ft-:..*-,.%,I 4-..,. I---.,....:.t.t..-1.,..-. .... ,"".....-....."'-,t, .... .e .,..'1`4`", 'mi-,.i'. ``. ,v,-,--v."',-,-- '. -, .; It ...L,''. 't :.....'.", -,.:. .,.- -S,4.1'.. - -j,,;. _....,_'. '. t, .h- 'il--.z.- .;.I ft..I.'...e.",-,-. -- -;, 7-. ."-; -1k,;'-t;';L 1%..;, --,? 1. .%-""I ; I ..,.! -;'--.. ,., .,.-4, "- " ..4 -4.-, L.:.-,1': ,,.- ,.- 'L _,j.?....-: 'O,-_., ,.,..1,,.,..!'..,.....I.,..". -.1% -.: :-,. ...-,.; ,; -..,.. jj.-' L" -14,.I,,Z;v1i,.3 -. 1. ....,;.;.. ,..,:,.,, ,I.-.-,.:,'.--...7Z %.,'t...I-., .. ,.',r!..iLI,;.--!,,-,..-. k .A, ,-..4, :! -.;,. .-.1.,...,.,x,.,.'A'-%--4.-.. ,.,,.....*W,, _-'.,.,-;t. ",J.'...,",..: .I:..'t.".1.'..1.L,.:..,--'.. .' """". .-,o'.-.%7: -1 1, !; ,.- .4 -.II-,...i!;.1 .I.,*L I,.. A,.. - ... .,.... - .A ..!L..-.,vi--.- , k. "..--,,- ..., ....-.. -1,.,- ...,:.-;.I..;.' .,:. '.", .. ...%i,,. .._.!t._'.11I.1.,-.,I.;..a, ,---.'.4...-,., tj. ,. ,;.-I&'* " *-`. .-'. -1, .4 ."..'.." .4_-..'. I-...-..1"L`_r, ., .'.:%.., ... .-4L -i4-Z.Z" . N.,',.'_,.,',-%-1..- i,."'..,- -..-..--...'. II.1 ,LI'%...... , ". ,,Z,,, -.'.4',-. -... "'iI_-1,,,..:"--t.1.1 -V.-4-1'. -.'.11 .I - L.'...L*T.,,.F - - .. .; !'.. -. I lz-.. ti_:,!, ."? "; ;1-.,;"t.., '!...-....I... .-..1_.-.,... ,.. .. '.,' -"? ",.,v-."'d.,'P., ;;.. ;L,: ...Ii-, ,I..:... ",-..,,..,...-....-...,"...I,--...;. -'.....1..., ,,-,..1 -: .,I, , . ,L ..",...,..,,.,f7., .. ',..7..,...,-.. .,-...I.4..11.,Z.1:.,. -I....., ., -1.,-...... I..,ZIt-V,.. -'Xeli__ 1". _,...... - ,. ;Ile..-- ....: ...., V.,Z.L.11..6 .. ,;..t.....I'. :..-. ._..-!...__ * ", ... 6.. ,t.-.-., ...-S, -.. .- -..-1, ...-ft.L'ilp--. .", .W ,-.:'.--,v.I.11': ,ir ".11 -, - -. -. ;,.. .-,'.' '. , .,.-.,-r ,-eI--...,1,-, , A,- .. -. 'Z -.,. -,:,.,.I..:, . ,4,: .;...... , -". ,;, ,,,. -.- -_ '. .1 .. A', I.i' 11. . " 1_*4..-,. -11z'V- .,. li .L - A-...4.1' -;,,..,....I;,;-I,, -- ."d',.. ."d.-". .j, - .. '. 4. I.. ..-.4 .- 'I, ,.,...,:-.;,.L,, ".".-..-- -,s-.'.; -, %,-. j. -..! 1j. ,. -LI,-ee,.'x.-'A. ,,, .. "..I--.- I7_.:'-., ..! 'I ... ,# ..- .I ", *., 41 " ':..'.-1... -i.-.;.';A..-I-....k.-I".-v ..,,.-,..,...".. .. I.", A-, 1.4 ,,.,.- L'-.,. 4- .., -.- "' -j. ,.,.,L.". -. ,. -, ,,-;... '.--'.4 ,,'.'.""..,.I..-' .-'..; .4 .1 ... ..- u*_r.,'If,_....,..;4..--.:....,.,"IT, '11.-.-..:--....,'t i. .1 I,1. . -., e. - -..- .1r. -,; .-.,. .--... , 't. ,.i %L :11 ,T", ."-- ...- -'_...'-. .!.-. ..."....'?.',-4.i.3 .4f "' -Z'&...,f,.'. ", -..'. .J'... ,4.-' ..,.lI.,'. ..,.:,tL. .,3.It,---.,".,!,4., .,j , ,..:-",..-i, ,.,,.`.,,,:, .. f...,... ;.,, - " ..i.. I..!.;,, :.', -.-.... -.-. .. t+ ."...C'..-.-.- - .., .. .,.-. ,,.,.-oI.e.LI-,%Aa.', -., .7 ._ 1,-I,-,..- -.!. Z ."" .,,,,;..;-__ 5;I, -1...t .,";"w..-.'%, .. -..', ,.,I-.., ". ...-.it ,.1,,'. _ .-.....-.. ., 6.-..,.,ji -$,-,z..... "!' .*. _.. 'JL "_-'" Z IX.,.&, 1,-;7 'G... ..,- -,,-`,-,:.-.I.., ,,,, -1 . "'I- "I.,.--.4.4,-.1,-..*,!4-,"-';.!.%-I'i-,:,.....;.-..I.---, 't- 77,;1'. ." -,-., 'aL;-. ... -; ts.,.....-,. .-. c-: ... -'.%> -...';."."- ':...... IN,1.'? ,.. -.,' ..,. j' . I-I,.,.---.",-, ..-..-,,e,,.'.m-,,..,If"-,`'-..,"..- ,,. .-.-. -., 'I..- ,. 1. '.. .,z. ,., ,; .11.*I ,.:.,. -,.., %.4'..--#,-- -,'., -..-,.,... , 4t'.-.. .Z.,,'. '.. .r.11,.,.-- I ji ."!.. '-,-.. ,1 .--";r-.4-. _- -"'.0 , ..C -. -,. ..,,.." 4-,t.,. ., .., -.---".,..,... -,I"`,, .....,. ,.. .--., .:, --!L. F.. ,., ..*..,..! . "., ,'- L-.,4,-,:,j:, -,..,r..!47,. Z- .., 4. 1 .....-.fl '., .., .,.,...... ,-,L:.:--., %".I'...'.--,.'41.-.'.. -I;. I--.. ..V,'. ".-1-1.1.-..-4-,;..-'.,...,"',-1. ,.,...... ,,kl,, .,.....,4L;.,:`rt....-...... ,,,..,...... 6'..-...!.-!-..:..,,.'..-,'?I-',,...,-1.-. ': .; 'i ." ": ,,,-.I..-. Nir, -- . ..'.,,'..I..0 ,_.. ,1.4.-C-.'-I...L. &_ -, ;4 "-, ,*.-.."4.-,-%I "I,-."C.-.-...-.., '. !L .. ,--.1 .v ,... ii....., . .V, -,I...-,...'t,.:.-. .-A4 '.jI-,X..,,;.1. .I .'..,1, ..,,,,.". ,... .. ,-- ,.. 1:-,.. .. I...p..&,I-,,....,-,.,_-; ;.. ,. ?;..,S.,,..4'..S -%., . , .. -.e A. ,,;.,I -. , .:;, ,w ,4 I'".4. "..; It.- ..".*Ii .-.N.,;A.%-,'.!..,: .,.-I-.fi*li.-,-,".,.!' j.-C.,' .,...4-,1,--:....I`:'.., 11,.,I-..,-i ..,."..;.'. .L_..;. .,., ... 1..,%,., .. -, i-I ---,,1. ".i_.,1k, .--..'. .,.. %,,-1,..-,...... ,.Z.,* ...,- ,:, '. ..,-"...... _ %, ..:...... 1..', ;.1-114 -.,., -- ,oI...-.;..:,-:-..:.,.,I.. .1:`.-.,..,.'.._..'.- ,-. .2.. -,-,. ;.,. ,'. Z.' ,7.. _L. .1, :.. ,.--- , ,.-'.....".e...1...." .. ...,,;4-..,..,-'i. V -. -., 11, .,- ..:.,,,"...'..;, - , tt -,.. ,.. I.'.-.1 .." . 74t,--. .; -. .-,<-.1 -a' %4.,- ,_-.o"', 4,1.- ., !. --L, -. -.i, ,_S '.."l, 1..". -,... .,-,,, '.,t'.;.,4!!.,z-% .j'. I , .-1 .-1,,-.". -" 1, '. , S, ;. ,. _.. P. .., -,'.:,'.;it.. .-;.P,0-.,_"I.:.,,...,-. .. .: 1; ,i-.,". ;. .L".,F .,.' 'r.,-"". z. ,..,...1.., .". .,,.'. --..,-,.,.-.,." ,..-'* -IrI.,.'i..!"., ,.-.`.1: 7 -%'....-, K ` , .;,,.!.'f -- . .; '? - -....a,. -%.. -."- .. -, -I'.- .I...... -IN .. ----, w. -i .- . ---II, ..... 'L-,-:--, r. ,t. .6 ....-,"...V. 1:r -,,.-... A..40..._--1...... iIL..1,:. 11..'...Jl. ... I--t-.. 'W - "t ... . .; ,., W .. ,...... A-.";,;...,.,?".,.;...... 4"-.,;. N. -...,-,J'-- .'. -.1.I., .". -% ,...-.'..* .. ,!1'_-,.;- _,..I-.,1: ." 'k .z. 4." ..,--,.'_. -, . .." L.- '.-#'.' j..,*...,'A C.,.,_-f,--.1 ,- .. ',.,i' -a I-.N...... ,e : -1 #, ...I.,I;:.", 7_4 ..'..'.. .;.J-.. .,Iif ,,".. I.' ....!7""....-i., "-, f, .''.I.. "_-:.4,M. ..' _., ,,,L .L. ,_ .. ..,S .. -M, -. .. .I ...L'i ,-- -.',.._r 11.'- .. t. --?.,.,. .,. -.,.....:.._,,.,`I.i".,;L _. "1`41,-.-..1."..!,--,. ... h.. V, ,.--.--, .i.,_,--.."-f.2. ,. ..,1. _'. ,_ ...1-1,-, :, .u."..,._-.,.--.,,.., >- ..I,*,A': -V, z_ .,,, -, -,,.'..'1'.-+ v-.,.--*.',..,. .11-1 i_ -.o,.. -_ "I--'k -., .!t .1' .. I...n."".....I.,-..,.. ;';. 41.,_'. :4 ...iA'.t.;;!..A*,,, ---- '- - P,- -i ";- ,4..1, - .,,. ...I'.;" ."L. ...!L -4 ?'-1k -q. i- ."...... -. 7 ,,,- -.---. -.'- -. -.,7Q 'I"-,.-..,;, -, -,., ". .- ,....-,....,1, ..,,--,: -5-;.;-, *- .;' -, ... z ,4 .,----.,, "-4:.%- "'...... -"i'",,, !. .q-!--- AV: ...,.,...I.21 .-, -.!, &",.- !._ !F.v.t*,,.i_-.. 1.,ih.J,.,t--- i.. -...'.-.-, -, ." I.I.._..;. V...,., I ....,".I"-...;,-II,;. .. ,. '. .. ..,;;;L .-..,- .%1. I..." -, L, !..* V.I,"-'...' ."! .4k', ..k,-V '. 'P`_' ...%..- .-,I.,-,-1.:, '- ".4,.., ...'. .?,.),.....'''.- --, -T --...L.,,.., ,; .L.. .'L .... ;:..,.,- .`_ .4e...I.-,:..;I----.''"4L' '__ ., ,,:1.,,'.. -,. W,,_%..,.'.....,..-*_t- _"...... L.,". %..O. 14, -4, .: .. - -0.: *-.,4.V,-Z'.,.. .,,_`..,,'.-I,,-,_i....., ,... r,,....-2, .-.-.-'r, .,. ^- -"t, ""' ,.'.I,... ,-'. '. '." L',.'.''' .-.,..,?--*-t ...-... ;,-.---I..-I - .,;ig- ... ,.j.,-- '..,'- -.. ,-. -,",,_-..4.. .-..--7 .-,-- ,-:",.. ." .4L t" --- ...46,.V.i.-,O'.-.....,I I. ., .'..!' ,,,,e-`-'..-aI.,-".- ...... t.---,!.-.'.. 111-`% 1'..-11_ ': ,. 17.,-I.I.,.,..!. .'_; ... ,. 1. _.,; ".; : '. !, .,.., "..,.1,. -., "-;i. 4..,W. j, ...,. ., ,".Ki-.' 4-,,..,..-...O%.r.-L,:,.';,..IF, ,-- ..--"--....:.,-, 1. -.-,.-4,-t.-:, ,v., --.%---I1%,..ts, J. ... ,%A,.. At7.t, -.1: .- -.-....._"'.",,:'.-...... I,..L..%I.. " -1 , 's, _.: ..-L'.. ...,..`.. -, ..,:, .. ," --:--:: .. ,,, .,--, ., i...'" 'L.t, v- I.."..-- "i .,- ' 7.....L#4..F". V "; - ..;,-*-.I...-, r. QV. ,6-,, ...L'... -!:! ..-.. -:-'r,'..-.,..--,.. ,;., *Z`:L t .4. ;, ." ';,, -- ,,, .-..11I..1I.",...i- .'-- - -- ; ,". %-_:.,I" L` 1,.',r ,WL.. I!t.-t ", N" ... '.," -%'. .,,II,-_,-.,- '.!, "r.-1- -4,Z,";..,."._, "',,.:, -.;,.. '-L.1"I'!.- --. ,..,v! -!- Q, ,.',-.%,-. -- .-.,-.- ..V .%,. .,:N-,L----.,"" ,-,.t >. 4,.,"%--'. '4 .. , ... ..,:V..._..-!,11.,....-...... --.. .; .." , -, .,.-._.4..-..,I,-.;.".v.- .1-"' 4.4 . .-.. .,-. .-,- -S-t- '.. 6 ,I. ._1. -"....'. ...'.r,%-...... ,1,

infectious; the quarter-length molecules were not. dicated that there was no residual virus in the preparation. The specific infectivity of the two batches of DNA were 1550 Replication of HSV after Transfection of Lymphoid Cells. PFU/,fg for batch 4 and 300 PFU/jug for batch 6 when 100 ng When a mixture of viral and carrier DNA, CaC12, and Hepes- of DNA was added per dish of BSC-1 cells. The calculation of buffered saline was added to lymphoid cells suspended in their Downloaded by guest on October 1, 2021 952 Medical Sciences: Miller et al. Proc. Natl. Acad. Sci. USA 76 (1979) in _ Exp.1 In the three experiments shown Fig. 3, samples of cells and 4.0I fluids were harvested daily and the numbers of PFU in frozen and thawed samples were titrated on BSC-1 cells. Virus was first 3.0 1~- detected at 48 hr after transfection in one experiment and at 72 hr in the other two. Maximal titers of about 105 PFU/ml 2.0 were reached after 4-6 days. The number of infected centers responsible for the viral 1.0 0to* growth curves was measured (Table 2). There was one infected <1.04 center per 5 X 104 cells at 24 hr after transfection of X25-9 cells. I T~~ I I Because there was no virus in the supernatant fluid at the time of appearance of these early infected centers, they represent the minimal number of cells that were transfected by DNA. In Exp. 2 * three other experiments (not shown) the number of cells initially 4.0I 0 transfected and competent to produce mature progeny was 0.5, E 3, and 5 per 105 cells exposed to DNA. Infectious virus was - 3.0)I found in the supernatant fluids of the X25-9 cultures 24 hr after the first infected centers were detected. Therefore, the number CL 2.011 of infected cells measured later in the experiment may result either from cells initially transfected by DNA in which virus .0 1.0 production was delayed or from cells infected by virus released <1.04 during the first round of replication. L~~I I1 I To compare the sensitivity of the X25-9 line to infection by virus and to transfection by DNA, both virus infection and transfection were done simultaneously under the conditions of 4:0 _ Exp. 3 calcium precipitation and Me2SO shock. Serial 1:10 dilutions of virus or DNA were added to individual cultures. Aliquots were harvested daily and tested for their content of infectious 3.0 / virus. The minimal infective dose of virus was 250 PFU; 25 PFU did not initiate infection. The minimal infective dose of DNA 2.0 / was 500 ng of batch 6, an amount equivalent to 150 PFU measured on BSC-1 cells. The virus growth curve after infection of the lymphoid cells with 250 PFU was similar to that seen < 1.0 9*4 after transfection with 1 ,gg of DNA (Fig. 3); however, a higher titer of virus (4 X 106 PFU/ml) was reached 4 days after in- 0 1 2 3 4 5 6 7 8 Days after transfection fection. Other Lymphoblastoid Lines. The X25-9 line has certain FIG. 3. Replication of HSV after transfection of line X25-9 with properties not seen in most other lymphoblastoid lines derived 1 Ag of HSV DNA. Three separate experiments are shown. In Exp. after transformation of umbilical cord cells by EBV. The cells 1 and Exp. 2, batch 4 DNA was used; in Exp. 3, batch 6 was used. are larger than normal and they are hyperdiploid: there are Samples (100 were collected daily from the culture and stored at Al) 70-90 chromosomes per cell due to triploidy or tetraploidy of -70'C. They were thawed, and plaque titrations of the mixture of some cells and fluids were performed on BSC-1 cells. many chromosomes. Furthermore, of the cells adhere to plastic or glass surfaces. Therefore, we examined two other lymphoblastoid cell lines from the same infant, X25-4 and usual growth medium, RPMI 1640, a dense precipitate formed X25-6, which have a normal chromosome number and which and was toxic to the lymphocytes. We thought that this pre- grow totally in suspension. Both the X25-4 and the X25-6 line cipitate might be due to the 5-fold higher concentration of were susceptible to transfection with 1 uig of HSV DNA (Table phosphate in RPMI medium than in Eagle's medium with 2). The time course of replication and amounts of virus pro- Earle's salts. Therefore, the latter medium was substituted. duced were similar to those seen with the X25-9 line. Successful Successful transfection then occurred in all successive trials with transfection also occurred with the X50-7 line from another the X25-9 cells. infant.

Table 2. Transfection of four different lymphoblastoid lines with HSV type 1 DNA Line X25-9 Line X25-4 Line X25-6 Line X50-7 Days after No. IC PFU/0.1 ml PFU/0.1 ml PFU/0.1 ml PFU/0.1 ml transfection per 104cells* supernatant supernatant supernatant supernatant 0 0 0 0 0 0 1 0.2 0 0 0 0 2 3.8 10 0 8 ND 3 20.0 30,000 43 50 ND 4 60.7 20,000 TNTC TNTC 28,000 5 69.0 50,000 ND ND 100,000 6 206 25,000 ND ND 900,000 Transfection was with 1 ,g of HSV-1 DNA, batch 6. TNTC, too numerous to count. ND, not done. * Infected centers (IC) were assayed as described in Table 1, except BSC-1 cells were used as indicator monolayers. Downloaded by guest on October 1, 2021 Medical Sciences: Miller et al. Proc. Natl. Acad. Sci. USA 76 (1979) 953

DISCUSSION HSV DNA. These events seem to enhance susceptibility of The present experiments were stimulated by the need to define umbilical cord leukocyte cultures to HSV infection (Table 1; precisely the conditions for consistent expression of viral DNA detailed studies will be published elsewhere). Assuming that introduced into lymphoid cells. We have shown that EBV- the techniques presented in this report will be applicable to transformed human lymphoblastoid cells of umbilical cord primary lymphocytes, several further studies can be contem- origin can regularly be transfected with HSV DNA by using plated. Because lymphoid lines exist that do not contain EBV, the calcium technique first described by Graham and van der it will be of interest to learn whether lymphoid cell immortal- Eb (9). According to one preliminary report, the Raji and Molt-4 ization can occur after delivery of specific DNA fragments from lines have also been transfected with HSV-1 DNA by means of other viruses such as HSV. It may now be possible to delineate the DEAE-dextran method, and the Raji cells formed a carrier portions of the EBV genome needed for immortalization, culture (12, 1). provided that a virion-associated polymerase is not needed for Several factors may have contributed to the success of these the infectivity of EBV DNA. Another objective would be to trials. One was the selection of a suitable cell/virus system. It learn whether the strict B cell tropism of EBV, which seems to was also important to use viral DNA of high specific infectivity; be determined by an interaction between the virus and a spe- thus, the full-length genomes obtained by gentle disruption of cific receptor on B lymphocytes, can be overcome by using EBV purified virions may have been especially suitable. Modification DNA (15). It would then be possible to learn whether the highly of the lymphocyte culture medium to avoid excessive precip- efficient transforming capacities of the EBV genome can be itation and damage to the cells appeared necessary for successful expressed in other hematopoietic or tissue cell types. transfection. In one experiment, 1000 PFU of HSV was in- cluded in the transfection mixture of Hepes buffer, CaCl2, and We are grateful for excellent technical assistance from Arlette Miller carrier DNA. Half of the inoculum was added to cells resus- and Elizabeth Grogan. This work was aided by grants from the pended in RPMI and half to cells in Eagle's medium. Infected Netherlands Organization for the Advancement of Pure Research centers (10/105 cells) were detected only in the culture in Ea- (ZWO) and the National Cancer Institute (CA 12055 and CA 16038). gle's medium. Toxicity of the Me2SO was minimized by di- G.M. is an investigator of the Howard Hughes Medical Institute. The luting it immediately after the treatment. The lymphocytes work was conducted during a period of support under the Macy Fac- were spread over a large area when they were exposed to DNA. ulty-Scholar Program. This allowed maximal contact of the viral DNA with the target cells without the need for vigorous shaking which might frac- ture the DNA. 1. Sheldrick, P., Laithier, M., Lando, D. & Ryhiner, M. L. (1973) The extension of transfection methods to human lymphocytes Proc. Natl. Acad. Sci. USA 70,3621-3625. will permit many new in the 2. Graham, F. L., Veldhuisen, G. & Wilkie, N. M. (1973) Nature experimental approaches study (London) New Biol. 245,265-266. of lymphotropic viruses. One area which can now be studied 3. Stow, N. D. & Wilkie, N. M. (1976) J. Gen. Virol. 33, 447- is the effect of superinfection of EBV genome carrier cells by 458. EBV DNA and specific EBV DNA fragments. For example, 4. Shope, T. C., Klein-Robbenhaar, J. & Miller, G. (1972) J. Infect. it may now be possible to define the region of the HR-I EBV Dis. 125, 542-544. genome that is responsible for early antigen expression (13). 5. Geelen, J. L. M. C., Walig, C., Wertheim, P. & van der Noordaa, When lymphoblastoid cells transformed by conditional lethal J. (1978) J. Virol. 26, 813-816. EBV mutants are available, it should be possible to perform 6. Klein, G., Lindahl, T., Jondal, M., Leibold, W., Menezes, J., classical marker rescue experiments with EBV DNA fragments. Nilsson, K. & Sundstrom, C. (1974) Proc. Nati. Acad. Sci. USA Conversion of EBV genome-negative lines to expression of EBV 71,3283-3286. nuclear antigens may be attempted with EBV DNA fragments, 7. Boyum, A. (1968) Scand. J. Clin. Invest., Suppl. 97, 21, 77- and thus the for these nuclear be 89. region coding antigens may 8. Henderson, E., Heston, L., Grogan, E. & Miller, G. (1978) J. Virol. defined directly (14). Because EBV is a plasmid, it has been 25,51-59. considered as a potential vehicle for cloning eukaryotic genes. 9. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, 456- Transfection of lymphoid cells is a step on the way to devel- 467. oping such technology. 10. Dean, J. H., Silva, J. S., McCoy, J. L., Leonard, C. M., Cannon, The availability of lymphoblastoid lines proved to be com- G. B. & Herberman, R. B. (1975) J. Immunol. 115, 1449- petent in transfection may aid in the cultivation of viruses that 1455. have resisted growth in vitro. 11. Henle, W., Henle, G. & zur Hausen, H. (1969) Cancer Res. 29, Some new investigations will require that primary lympho- 489-494. cytes, which have not been first transformed by EBV, be shown 12. Seigneurin, J. M., Desgranges, C., Lavoue, M. F. & de The, G. to be susceptible to DNA. It seems likely that stimulation of (1976) J. Immunol. 117,950-952. 13. Henle, G., Henle, W., Zajac, B., Pearson, G., Waubke, R. & Scriba, cellular DNA synthesis or cellular activation will be necessary M. (1970) Science 169, 188-190. to demonstrate transfection of primary lymphoid cells with 14. Klein, G. (1975) Cold Spring Harbor Symp. Quant. Biol. 39, 783-790. iDesgranges, C., Laithier, M. & Sheldrick, P. (1975) Third Interna- 15. Yefenof, E., Klein, G., Jondal, M. & Oldstone, M. B. A. (1976) Int. tional Congress of Virology, Madrid, Spain, p. 165 (abstr.). J. Cancer 17,693-700. Downloaded by guest on October 1, 2021