VOLUME 24 NUMBER 4 FEBRUARY 15, 1996
`
`Nucleic Acids
`Research
`
`
`
`OXFORD UNIVERSITY PRESS
`
`1048 Codm NARHAD
`
`Pfizer EX. 1019
`
`Page 1 of 10
`
`Pfizer Ex. 1019
`Page 1 of 10
`
`

`

`""c_'_"'.'_
`Nucleic Acids
`Research
`
`
`
`Executive editors
`
`R.T.Walkcr. Birmingham. UK
`R.J.Rohcrls. Bereriy. MA.
`(.iSA
`K.Calame. New l’ht‘k. NY: USA
`|.C.Eperon. L{’i(‘{.’.\'i£’i1 UK
`
`M.J.Gaii. Cambridge. UK
`H.J.Gross. Wiit'3bttrg. Germany
`R.I.Gumpon. Urbana. iL. USA
`
`R.B.Hallick. Tucson. AZ. USA
`S.Linn. Berkeiey. CA. USA
`R.T.Simpson. University Park. PA, USA
`
`Editorial board
`
`Afiuiroch. Genera. .S'n‘itzeriattti
`M.Bcalo. Marintt'g. Germany
`M.Be|ihri. Aii‘nm}; NY.
`i.-’SA
`M.Busslinger. Vienna. Austria
`H.Cedur. Jerusalem.
`ist‘ttei'
`
`RDalgleish. Len-ester: UK
`M.L.Di:P:1mphili1-..
`.-'\t'tttie\'. NJ. USA
`WDynan. Hattider. CO. USA
`REckslcin. Ct‘ittingen. Germany
`J.D.Engel. iz'i'ttnstnn.
`it'_. USA
`RTEngleLl. Httititntn'e. MD. USA
`D..I.Finnegtin. Etitnintrgit. UK
`M.Ge|lert. Bethesda. MD. USA
`R.Giegé. Strasbtnnjq. Frant'e
`LIAGI'lViill. Atttstet‘tittnt.
`‘i‘t'te Netiterittntis
`WGruissem. Berkeley. CA. {.-'SA
`W.Guhchlbuucr. (iiiLsttt'-l’-.-ette. Franee
`S.E.Hali'ord. iiristai. UK
`D.HaWIey. Eugene. OR. USA
`
`J.E.chtst. Berkeiey. CA. USA
`CHéléne. Paris. France
`A.J.Jeiireys. Utieester. UK
`S.-H.Kim. Bet‘keier. CA. USA
`A.R.Krainer. CSHL. NY. USA
`R.Krumlaul‘. London. UK
`A.Lamond. Dundee. UK
`D.S.Lalchmam. Landon. UK
`C.J.Lcavcr. Oxford. UK
`D.M.J.Lilley. Dundee. UK
`J.T.L.i.~;. ititttt'tt. NY. USA
`R.Luhrmiln. Mat-intrg. German}-
`N.C.Murlin. LilitiSl'iUt’. KY. USA
`M.McClclland. San Diego. CA. USA
`WRMcClure. Piti.\'hitt'_t:it. Hi. USA
`M.Muramatsu. Saittttna. Japan
`E.Oht§uka. Sapporo. Japan
`H.0kaytlma. Tithe. Japan
`M.V.Olson, Seattle. WA. USA
`
`M.G.Parkcr. London. UK
`R.K.Paticnt. London. UK
`MPaule. Ftn‘t C(tiiinx. CO. USA
`A.M.Pyle. New York. NY. USA
`J.M.Rosen. Houston. TX. USA
`H.Ruterjans. Ft'fltiKfiii‘i. Germany
`C.W.Schmid. Davis. CA. USA
`P.M.Sharp. Nottingham. UK
`l..Simpson. Lax Angeies. CA. USA
`S.T.Smulc. L115 Angela’s. CA. USA
`G.Stonno. Seattie. WA. USA
`R.H.Symons. Adelaide. Attstrait‘a
`K.Tuiru. Isakttba. Japan
`[Tinncu Jr. Berkeie)‘, CA. USA
`B.Vun News. Minneapolis. MN. USA
`A.M.Weiner. New Haven. CT: USA
`S.(‘.West. Sntttit Mimtns. Het'ts, UK
`J.A.Wise. Ciel-Timid. OH. USA
`Glut}. Faster Cit): CA. USA
`
`Editorial and Production
`
`Joy Walker. UK Etit'ttn'iai 0 11)
`Carol Cook. US Edittn'iai ()fi‘iee
`
`Sarah Brennan. Pmtittetimt Editor
`Jaquelinc Gruingcr. Ptrta‘nctian Assistant
`Robert Blundell. Prniitn-tiatt Assistant
`
`
`
`Pfizer EX. 1619
`
`Page 2 of 10
`
`Pfizer Ex. 1019
`Page 2 of 10
`
`

`

`/’—————__—/——
`
`Nucleic Acids Research
`
`_/_/———————
`Contents
`Volume 24 number 4 February 15. 1996
`///-
`
`HNA
`
`Base and sugar requirements for RNA cleavage of
`mential nucleoside residues in internal loop B of
`the hairpin ribozyme: implications for secondary
`structure
`
`S.Schmidt. LBeigclman. A.Korpeisky. N.Usman.
`U.S.Snrenscn and M.J.Gai1
`
`Preparation of biologicaily active Ascot-is strum
`mitochondrial IRNAMet with a TV-replacement
`loop by ligation of chemically synthesized RNA
`fragments
`T.0htsuki. G.Kawai. Y.Wat:tnabe. K.Kita.
`K.Nishikawa and K‘Watanahe
`
`Structural characterization of U*-l915 in domain
`W from Escherichia coii 235 ribosomal RNA as
`3-methylpseudouridine
`J.A.Kowulak. fiBrucnch T.Hashir.umc. LMPEIItflZ
`1.0fcngalnd and J.A.Mc("|oskcy
`
`MOLECULAR BIOLOGY
`
`Inducible site-directed recombination in mouse
`embryonic stem cells
`YZhang (‘Ricstcren A.—M.Ayrall. ESablilzky.
`T.D.L1ltlcwuud and MRth
`
`Activation domains of transcription factors
`mediate replication dependent transcription from
`a minimal HIV-l promo“?
`R.D.Williums. B.A.Lec. SRJackson and
`NJPmucll'tml
`
`Binding to the yeast Swi4.6-dependent cell cycle
`'3‘”. CACGAAA. is cell cycle regulated in NW!
`L.A.Harrington and BJAndrews
`
`DiiTerential transcriptional regulation of the
`3PM! gene by retinoic acid receptor homo- and
`heterodimers in yeast
`AJSnlerno. Z.Hc. Aflous-NilssOR. H-Ahma and
`P-Mak
`
`The DNA-binding protein 11de (a putative K"
`homologue) is required for maintaining norml
`tel
`.
`m as “WNW“
`omere length m Saccfingfiitchie and T.D.Pet95
`S.E.Porter, P,W.Greenw€
`
`Elements within the B-lactoglobuiill gene inhibit
`e
`albumin cDNA and
`x.Pr'a‘si‘ion of human serumls but rescue their.
`e"dilatation in the "summary gland of transgenic
`mice
`.
`LBarash. M.Nalhan. R.Kari. N.Ilan. M5118?“ and
`D.R.l-lurwitz
`
`573—58 I
`
`Characterisation of intronic uridine-rich sequence
`elements acting as possible targets for nuclear
`proteins during pre-mRNA splicing in Nicotiana
`plumbagt‘nifoiio
`M.Gniadkowski. M.Hemmings-Mieszczak, U.Klahre.
`H.-X.Liu and W.Filipowicz
`
`662-667
`
`Mismatch DNA recognition protein from an
`extremely thermophilic bacterium. Themes
`thermophiitts H38
`S.Takumatsu. R.Kato and S.Kuramitsu
`
`Sequences homologous to yeast mitochondrial and
`bacteriophage T3 and T7 RNA polymerases are
`widespread throughout the eokaryotic lineage
`N.Cennakian. TMJkeda. R.Cedergren and
`M.W.Gray
`
`688—693
`
`543—543
`
`549—557
`
`558—565
`
`566—572
`
`582-2585
`
`602—6 l [)
`
`Artificial linear mini-chromosomes for
`Trypanosonm bracei
`P.K.Patnaik. N.Axelrod. L.H.T.Van der Ploeg and
`G.A.M.Cross
`
`A novel type of retinoic acid response element in
`the second intron of the mouse HZK" gene is
`activated by the RARIRXR heterodimer
`RJansn and J.Forcjt
`
`Atomic force microscopy ol‘ long and short
`double-stranded. single-stranded and triple-
`stranded nucleic acids
`H,G.Hansma. LRevenko. K.Kim and D.E.Lancy
`
`Multiple mechanisms may contribute to the
`cellular anti-adhesive effects of phosphorothioate
`oligodeoxynucleotides
`Z.Khaled. LBcnimelskuya. RZeltscr. TKhan.
`H.W.Shnnna. R.Narayunan and C.A.Stcin
`
`The influence of a tGTizq microsatellite sequence
`on homologous recombination in the hamster
`adenine phosphoribosyltransrerase gene
`R.G.Sargcnt. R.V.Mcrrihew. R.Naim. GAdair.
`MMeulh and J.H.Wilson
`
`Binding site analysis of c-Myb: screening of
`potential binding sites by using the mutation
`matrix derived from systematic binding affinity
`measurements
`
`Q.—L.Deng. SJshii and A.Sarai
`
`GENOME STRUCTURE AND MAPPING
`Sequence analysis of 56 kb from the genome of
`the bacterium Mycopiasmo pneumoniae
`comprising the dnaA region1 the atp operon and a
`cluster of ribosomal protein genes
`HHilbert. R,Himmelreich. HPlagens and
`R.Hernnann
`
`619—627
`
`640—647
`
`648—654
`
`668—675
`
`694—701
`
`'1‘] 3—720
`
`737-445
`
`746—753
`
`766-774
`
`623—639
`
`Pfizer EX. 1019
`
`Page 3 of 10
`
`Pfizer Ex. 1019
`Page 3 of 10
`
`

`

`—_'____——__—.____——-———————-\
`contents [Continuedl
`_____—_—______________—_—————————-——_______
`
`Volume 24 number 4 February 15. 1995
`
`ENZVMOLOGY
`
`Recognition of DNA insertionideletion mismatches
`by an activity in Socchoromyces cerevisme
`.l J .Miret. B.O.Parlter and R.S.Lahue
`
`CHEMISTRY
`
`Acid-induced exchange of the imino proton in
`G-C pairs
`S.Nonin. J.-L.Leroy and M.Guéron
`
`Hybridization properties of oligodeoxynucleotide
`pairs bridged by polyarginine peptides
`Z.Wei‘ C.—H.Tung. TZhu. W.A.Dickerhof.
`KJ.Breslauer. D.E.Georgopoulos. M.J.Leihowitr. and
`S.Stcin
`
`Rapid and eliicient hybridization-triggered
`crosslinking within a DNA duplex by an
`oligodeoxyribonucleotide bearing a conjugated
`cyclopropapyrroloindole
`E.A.Lukhtanov. M.A.Podyminogin. 1.V.Kutyavin.
`R.B.Meyer.]r and H.B.Gamper
`
`Use of a high affinity DNA ligand in flow
`cytometry
`K,A.Davis. B.Abrams. ‘1’.Lin and SiDJayasena
`
`The thermal stability ol’ DNA fragments with
`tandem mismatches at a dtCXYGl-dlCY’X'G] site
`S.-H.Ke and R,M.Wartcl|
`
`Triplex formation by a psoralen-conjugated
`oligodeoxyribonucleotide containing the base
`analog 8-oxo-adenine
`
`P.S.Milleri G.Bi. S.A.Kipp. V.Fok and R.K.DeLong
`
`Synthesis and hydrolysis of
`oligodeoxyribonucleotides containing
`Z-aminopurine
`J.Fujimolo. Z.Nuesca. MMaaurek and L.C.Sowers
`
`Antisense oligonucleotides containing an internal,
`non-nucleotide-based linker promote site-specific
`cleavage of RNA
`M.A.Reynolds. T.A,Beck. P.B.Say. D.A.Schwanz.
`B.P,Dwyer, WJDaily. M.M.Vagheli. M.D.Meltier,
`R.E.Klem and L.J.Arnold.lr
`
`STRUCTURAL BIOLOGY
`
`High-resolution NMR study of a GdAGA
`tetranucleotide loop that is an improved substrate
`for ricin. a cytotoxic plant protein
`M.Orita. ENishikaWa. "I“.Kohno. TSenda, Y.Milsui.
`Y.Endo. K.Taira and S.Nishikawa
`
`775—781
`
`flirt—1'02
`
`596—601
`
`193—314
`
`795—?96
`
`797—qu
`
`ill ill-lit] |
`
`Hill
`
`72 l—729
`
`586—595
`
`655—6“
`
`683—687
`
`"till—Tilt)
`
`"lll'l—T l 2
`
`730—736
`
`Structure and dynamics of the DNA hairpins
`formed by tandemly repeated CTG triplets
`associated with myotonic dystrophy
`S.V.S.Mariappan. A.E.Garcin and (itiupm
`
`Solution structures of the individual single strands
`of the fragile X DNA triplets tGCCi,,-l(i(iCl,,
`S.V.S.Mariappan. RCalasli. X.Chen. R.Rallil'l'.
`R.K.Moyzis. E.M.Bradbur_v and G.Ciupla
`
`METHODS
`
`Transfecting mammalian cells: optimization of
`critical parameters aifectint: calcium-phosphate
`precipitate formation
`MJordan. A.St‘liallhorn and l".M.Wurm
`
`Non-radioisotopic differential display method to
`directly visualize and amplify differential bands
`on nylon membrane
`l.l.W.Chcn and KPeck
`
`A novel ligation mediated-PL‘R based strategy for
`construction of subtraction libraries from limiting
`amounts ol‘ mRNA
`5.Ghosh
`
`Efficient isolation of differentially expressed genes
`by means of a newly established method. 'ESD'
`YSUZURL N.Sato, Mfl‘uhyama. A.Wanalta and
`T.Takagi
`
`Increased cloning efficiency by temperature-cycle
`ligation
`A.H.Lultd. MDuch and F.S.Podcrscn
`
`iii-1459
`
`Corrigendum
`
`Author index
`
`Forthcoming events
`
`"loll—765
`
`fill—(illi
`
`Neornycin, spermine and hexaamminecobaltilli)
`share common structural motifs in converting
`- to A-DNA
`
`676—682
`
`H.Robinson and A.H.—J.Wang
`
`Pfizer EX. 1019
`
`Page 4 of 10
`
`Pfizer Ex. 1019
`Page 4 of 10
`
`

`

`596-60!
`
`Nttcieic Acids Research. i996. Vol. 24. No. 4
`timization of critical
`-
`-
`'
`Transfectrng mammalran cells. op
`_
`_
`parameters affecting calcrum-phosphate precrprtate
`
`© two Uijflrr'ri Unit-emf). Pith
`
`
`
`._..__._.....
`
`formation
`Martin Jordan, Annette Schallhorn+ and Florian M. Wurm"
`
`Genentech lnc.. South San Francisco. CA 94080. USA
`
`Received November 8. 1995: Revised and Accepted January 2. 1995
`
`ABSTRACT
`
`co—precipitates arise
`DNA-calcium phosphate
`spontaneoustv in supersaturated solutions. Highly
`effective precipitates for transfection purposes,
`however, can be generated only in a very narrow range
`of physico—chemical conditions that control
`the
`initiation and growth of precipitate complexes. The
`concentrations of calcium and phosphate are the main
`factors influencing characteristics of the precipitate
`complex. but other parameters, such as temperature,
`DNA concentration and reaction time are important as
`well. An example for this is the finding that almost all
`of the soluble DNA in the reaction mix can be bound
`into an insoluble complex with calcium phosphate in
`<1 min. Extending the reaction time to 20 min results
`in aggregation andror growth of particles and reduces
`the level of expression. With improved protocols we
`gained better reproducibility and higher efficiencies
`both for transient and for stable transfections. Up to
`60% of cells stained positive for [i-gal and transient
`production of secreted proteins was improved 5- to
`10-fold over results seen with transfections using
`standard procedures. Similar improvements in
`efficiency (number of recombinant cell colonies)
`were observed with stable transfections, using
`co—transfected marker plasmids for selection. Transient
`expression levels 2 days after DNA transfer and titers
`obtained from stable cell lines, emerging weeks later,
`showed strong correlation.
`
`INTRODUCTION
`
`Coprecipirates composed of ‘calcium phosphate' {Itydrtis)-apatitei
`and purified DNA have been used for >20 years for the transfer
`to and expression of genetic inl'omtation in mammalian cells in
`culture ( l J. This technique has become one of the major methods
`for DNA transfer to mammalian cells. A number of papers (2-6}
`have. addressed observed variability ol‘ DNA transfer and low
`efficrency and they usually contain specific recommendations for
`
`‘optim'dl' pmcgdtnes. However. these cortuttuttications did not
`report on the complex relationship3i between different “WP"-
`ncnts responsible for the creation of a supersaturated status. We
`present here data that provide the basis for u better understanding.
`at the physico-chemical level. of some of the most crucial aspects
`concerning the t'orrttation of DNA containing precipitate cum-
`pleses. We have applied gained insights in sets 02' transfeciiortt
`both for transient and stable expression of recombinant proteins. We
`used two popular immortalized ceil lines I'or these studies: human
`embryo kidney 293 cells [HI'iK-JUM t‘f—‘JI and dihytlrofulrrte
`rcductasc—minus t[)HFR«i Chinese hamster ovary cells tCI-lU]
`llU—lfil. We were able to improve the efficiency ol'DNA transfer
`and expression in both cell lines.
`This paper also illustrates that a simple cenli'il‘trgation—OD assay
`can be useful in studying kinetics and reproducibility of precipitate
`I‘umtation. This test provides information on the reliability of
`precipitate formation and gives greater assurance for successful
`DNA translcr to cells.
`
`-
`
`'
`
`MATERIALS AND METHODS
`
`Plasmid isolation
`
`Propagation til AmpR plasmids were thine in the bacterial filial"
`DHirt Uslttg :‘tll itgrrnl carbenicillin in Lli rncditrtn. DNA ““35
`isolated using the alkaline lysis method and was purified by dwbl‘.
`equilibrium centrit‘trgation in (\(I'I—etltidittnt bromide gradient“
`described by Sztrttbr‘ook er (ii. [23].
`
`Calcium phosphate—DNA ctr-precipitation
`A Stilutitm of IOU ul of 2.5 M C‘aC‘Ig and the desired amount of
`Plasmid DNA was diluted with tilt) TE buffer (I tnM Tris-HG-
`[).l rnM EDTA. pH to; to a final volume of l tnl. One volume
`0i this 2X Car'DNA solution was added quickly to all equal
`volume of 2x HEPES solution:
`l4lJ mM NaCl,
`5.5 in”
`NagHP04. it) mid HEPES. pH 7.05 at 23°C. or alternattvcli'
`PhUSPhatc-free 2x HEPES buffer was prepared and supple”
`ented with phosphate from a 300 mM stock solution of PH 7‘95
`containing 195 mM NazHP04 and 105 mM NuH1p04- Wm“
`
`___________..-
`
`.
`.
`at
`.
`.
`1"To whum correspondence should be addressed
`Switzerland
`prawn] mum” Delmmm “f Chem-“"3- SWISS Federal Institute of Technology Lausanne. "315 mum
`‘
`.
`.
`.
`+
`.
`Present address. FH Mannheim. Hochsehule l'iir Technik und Gestaltung caret Mannheim Ge
`.
`.
`. mtany
`
`Pfizer EX. 1019 '
`
`Page 5 of 10
`
`Pfizer Ex. 1019
`Page 5 of 10
`
`

`

`Nucleic Acids Research. l996. Vol. 24. No. 4
`
`597
`
`'30
`50
`50
`4|}
`30
`20
`10
`
`2000
`
`1500
`
`1000
`
`500
`
`b “
`
`cells
`
`it.positive
`TNK—tPAinnng
`
`1 min.
`
`5 min.
`
`20 min.
`
`1 min.
`
`5 min.
`
`20 min.
`
`Figure 1. Transient expression oi'B-gal or "WK—[PA in HEK—293 and in CH0
`cells transfectcd with a calcium phosphate-DNA precipitate complex. The
`precipitate was allowed to from for l. 5 or 2i] rrrin after mix ing and then added
`to the cells. tAi Cells were l‘ixcd and stained with X—gal 24 h after transfection.
`[Bl Supcrttalrlnt was harvested and analyzed after 6 days.
`
`RESULTS
`
`Complete DNA binding can be achieved within seconds
`upon initiation of crystal formation
`
`Subsequent to the mixing of the calciumiDNA solution with the
`HEPESiphosphatc solution a slight opacity appears within a few
`rninrrtes. indicating that a precipitate has been formed. With the
`intention of having the reaction 'complete'. standard protocols
`suggest an incubation period of up to 20 {4) or even 30 min t 3)
`at room temperature. To measure the rate at which the DNA is
`being incorporated into or associating with the fonning precipitate
`an assay was developed. This test was based on a centrifugation
`step. The DNA concentration remaining in the clarified solutions
`was assessed by determining the optical density at 260 nm.
`Surprisingly. at a pH ol‘ltlfi and a DNA concentration of 25 rag/ml.
`adsorption of DNA occurred within 30 s.
`In fact 30 s was the
`shortest period for completion of this test. When either calcium
`or phosphate was missing in the mixture. no DNA was found to
`be adsorbed [data not shown).
`
`solutions were mixed quickly once. and added to the cell culture
`medium after the time frame indicated.
`
`Ccntrifugation assay
`
`Precipitate formation was confirmed and quantified by absorption
`at 320 nm against a blank lacking the phosphate or alternatively
`lacking both. DNA and phosphate. The association (binding) of
`DNA with precipitate was determined by OD measurement at
`360 and 320 nm of a 250 pl aliquot of the supernatant of the
`precipitation mixture after 30 s centrifugation (16 000 g} in an
`Eppendort' centrifuge. The supernatant was analyzed immediately
`after the centrifugation step. Precautions were undertaken to
`prevent heating up of the rotor when multiple samples were
`analyzed. For precipitation experiments at 0°C the rotor was
`cooled down on ice before it was used.
`
`Cell culture
`
`Cell culture was performed according to Doyle er of. [21}. Both
`CHO cells and 293 cells were grown in a DMEMiFlZ l:l based
`medium. supplemented with 2% fetal call'scrum. CHO cells were
`maintained attached in “i5 cnt2 ilasks. Cells (293 HEK) were
`adapted to growth in suspension and grown in 250 rrrl spinner
`llasks. Both cell lines were subcultivated once or twice a week at
`ratios between HO and 1:100.
`
`Transicnl transt'ections
`
`Cells from the ex porrcntial L—‘rowth phase were seeded t 1—4 x [05
`cellsi‘mli
`into 12-well or fit} nmr plates the day before the
`transfection was done. One hour before the precipitate was added.
`the medium was replaced With l‘rcs'h medium (pH 14}. For each
`ml of medium. 100 pl of precipitate was added. With a calcium
`concentration of 125 mM in the precipitation mixture.
`the
`resulting final concentration ot'calciunr in the cell culture medium
`was ~ I 3.5 mM. The cells were exposed to the precipitate for 2—6 b
`all 37 'C' at a pH of 7.3—7.6. For CHO cells. a glycerol shock was
`applied at this point. The cells were exposed to 2(1'7r glycerol in
`PBS. After l min the glycerol was removed by adding fresh
`medium. aspiration of the mixture and replacement with fresh
`medium. For 293 cells the medium was replaced by fresh medium
`without applying a shock. The cells were then incubated for 1—6
`d3.“ before the supernatant was harvested and analyzed by
`ELISA.
`’l‘ransl'ectjon efficiency was determined by staining
`B-galactosidasc expressing cells with X-Gal after 3-4 It (22).
`
`Stable transfection for CHO cells
`
`A 1: l (wtwi mixture of linearized and purified plasmid containing
`DHFR as a selective marker or the TNK—tPA (lo) expression
`cassette was precipitated as described above. The cells were
`grown for 2 days after transt‘ection in non-selective medium and
`the supernatant was harvested to measure transient expression.
`The cells were trypsinizod and seeded in 100 mm plates under
`different selective pressure. Of these cells, 2% were seeded in 100
`mm plates
`in medium lacking glycine. hypoxanthine and
`lhymidine {GHT— medium), 20% in GHT— medium containing
`30 nM methotrexate (MTX) and 40% at 100 nM MTX. After 10
`dill/5. the product titers from pools ofcells were assayed by ELISA.
`For each level of selective pressure. titers seen from emerging
`Pools of stable cells were normalized against each other.
`
`Time-dependent changes in the precipitation complex
`that atfect the efficiency for transfections
`
`To determine the optimal incubation time for the formation of an
`etl'rciettt precipitate for o‘rmsfections. the reaction mix was trans-
`ferred to cells at dilierent time poims upon initiation of the reaction.
`A B-galactosidase (figul) plasmid ([4) was used to estimate the
`number of tnuist'ected cells (Wansfection efficiency]. An expression
`vector for human tissue plasminogen activator [TNK-IPA) (15.16)
`was used in a second transfection experiment.
`Highest translection elflciencies were observed with precipitation
`mixtures that had been incubated for short periods of time. [n a
`representative experiment ~60% of CHO cells and 38% of
`
`Pfizer EX. 1019
`
`Page 6 of 10
`
`Pfizer Ex. 1019
`Page 6 of 10
`
`

`

`598
`
`Nucleic Acids Research, 1996. Vol. 24. No. 4
`
`lb
`
`
`
`Figure 2. CHDceIIs with precipitates. visualized by phase-contrast microscopy
`(430x magnification] 4 h after adding the transfection cocktail to the cells. The
`precipitate mix was incubated for till I. (Bi 5 ortCl 40 min before transfer to
`die cells.
`
`HEK-293 cells stained positive for B-galactosidase in plates
`which had been exposed to l min precipitate complexes (Fig. IA).
`The transfection efficiency decreased to 3-5‘% when precipitates
`were used after an incubation period of 20 min. Using a 5 min
`reaction time gave intermediate resuits. Similar dil’threttces. here in
`yield of a secreted protein. were observed with a transl'osted
`TNK—tPA vector ( Fig. I B ]. Almost 2 pgftnl for HER-293 cells and
`0.5 ugl'ml for CHO cells of TNK-tPA were detected by ELISA in
`the supernatant of cells exposed to an 'early' precipitate—complex
`(l min]. ‘Late’ precipitates (20 min] gave only 10% of these titers.
`confirming observations made by O‘Mahoney er al. (6].
`These differences in transfection efficiency or in the levels of
`secreted recombinant protein could be correlated with the nature
`ofthecalcium phosphate precipitate in plates (Fig. 2). Precipitates
`added [ min after mixing. consisted of a large number of very small
`particles covering the surface of individual cells almost completely.
`Many particles scented to adhere to the cells. however many were
`floating in the medium and exhibited Brownian motionThe large
`number of particles and. possibly. Brownitut motion may be
`responsible for the image being of poor quality. Precipitate formed
`within a 5 min reaction time consisted of fewer but larger particles.
`None of these particles were floating. After 40 min the precipitate
`consisted of even fewer particles. some of them as big as the cells
`themselves. The highest nansfection efficiency correlated with the
`generation of many very small particles.
`
`Factors affecting the kinetics of precipitate formation
`
`Multiple aaitsfections. done on the same day with the same solutions
`usually give reproducible results but the transfoction efficiency can
`vary diarrtatically if experiments are performed on different days
`(own observations and personal communications). This provided
`
` boundDNA(ugrrnlt
`
`188mm Ca
`
`250mM Ca
`
`boundDNA(norm!)
`
`125mM Ca
`
`Figure 3. DNA binding capacity of a forming calcium phosphate precipitati- .1:
`1.5 and 20 mitt. {At Increasing DNA conccntnttious from 25 to fit} pglml at I33
`mM calcium.
`IBI Precipitation of 50 pghnl DNA at dili'crcrtt calcium
`concentrations.
`
`the motivation to systematically search for parameters which affect
`the precipitate formation and would change the efficacy in
`transt'cctions. Chen and co-authors (5] reported that lltc plasmid
`concentration needs to be optimized to achieve high tt‘anst‘ection
`efficiencies. We verified this observation and found that
`the
`
`amount of DNA can have a major effect on the precipitation
`reaction (Fig. 3A). At 25 uglntl all the DNA was bound to the
`limiting precipitate within l min. Higher concentrations ol‘DNA
`pautially inhibited the l‘orittation of precipitates.
`resulting in
`reduced amounts of DNA being associated witlt an insoluble
`precipitate complex. A DNA concentration of St] pgr’tttl DNA
`almost completely blocked the formation of precipitates. Even
`after a 20 min incubation time. (209’: of the DNA was associated
`
`with a precipitate.
`In an independent experiment (no temperature control] we
`show that increasing the calcium concentration could reverse ”“5
`phenomenon. 125 tnM calcium (standard) was compared wilh
`calcium concentrations of up to 250 ntM tFig. 38]. At a
`concentration of 2-50 mM calcium twice the amount of DNA was
`
`transferred into an insoluble precipitate. almost as last as seen
`under the standard calcium concentration for 25 (Lg/ml DNA.
`These data indicate that DNA participates early in the prom-‘\
`“lPTCCiPilale complex formation. The interplay ol'concentrationt
`ofcalcium and DNA points to the possibility that DNA molecules
`may ali'ect early events during nucleation and ‘ct'ystal' growth.
`Another important parameter is the temperature. DNA bindntg
`experiments were performed with a HEPES-phosphatc bufferlll
`“’hiCh “75 PhOSPhate concentration was reduced from 0.75 In
`0.6 mM. Results of one of these experiments are presented here
`(Fig. 41 Under reduced phosphate concentration the rate or
`PFCCitJitaIe formation was significantly slower. The indiVldfa'
`reactions Were performed at temperatures ranging from 0 to 37 C-
`At 20°C, a l min reaction time was no longer sufficient to
`completely bind the DNA in solution. Even after 20 min unit
`40% of the DNA was associated with a precipitate. N 3
`temperature of 31°C, however, fast and complete removal elm?ll
`from the solution occurred during the centrifugation step. No C
`
`Pfizer EX. 1019
`
`Page 7 of 10
`
`Pfizer Ex. 1019
`Page 7 of 10
`
`

`

`Nucleic Acids Research. 1996. Vat. 24. No. 4
`
`599
`
`A 50
`40
`
`20
`
`
`
`E an
`
`O E
`
`3O
`
`.D
`Ca
`‘5m1
`
`mtvt or phosphate
`
`Figure 5. Effect of calcium and phosphate concentration on the precipitation
`of DNA :5“ 1.1g DNNmt. ETC. pH 105. reaction time = l mini. Five calcium
`concentrations. ranging from |2.5 to 350 mM were tested. as indicated in the
`figure inserts. on Soluble DNA in the supernatant as a function of the phosphate
`mncclttrtttinn. [Bl Turbidity of the precipitation mixture Isuspensiont measured
`at no not.
`
`'—
`'
`. y=15.B XW'M’ 3:0.992 i
`
`3.5
`
`It:
`3
`to
`E 2.5
`8I:
`2
`a
`1 5
`.5
`.
`5
`1
`E
`0.5
`
`
`
`on
`
`so
`
`too
`
`150
`mM of calcium
`
`200
`
`250
`
`300
`
`Figure 6. Relalronslnp between calcrunt and phosphate concentrations
`sufficient to precipitate fill"? of the IJNA tt'tlhilt l min at 3.1 "C in atsolution with
`tit} pgjnd DNA at pH ltlfi.
`
`l35 mM calcium): a reduction of phosphate by
`precipitate tat
`35% to 0.5l ntM completely prevented binding of DNA.
`[a Figure 6 conditions :u’e summarized which address incom-
`plete binding of DNA. Concentrations of calcium and phosphate
`that will precipitate and bind 50% of the DNA provided were
`calculated from the data of Figure 5A. These data show that the
`association of DNA with an emerging calcium phosphate precipi-
`tate is a function of bodt calcium and phosphate concentration.
`
`Correlation between precipitated DNA and levels of
`expression
`
`Using the phosphate concentration as a tool to change the nature
`of the precipitate, we designed an experiment to conelate the optical
`characteristics with the uanstiection efficiency. Sufficient volumes of
`solutions were mixed to quantify the precipitation step and also to
`
`Pfizer EX. 1019
`
`Page 8 of 10
`
`boundDNAtpgt'ml) 24C
`
`Developing an assay to quantify the precipitation step
`
`Spontaneous precipitation occurs only if Concentrations of
`calcium turd phosphate are h i gh enough to ensure supersaturation.
`r’tll tlte data presented above suggest that conditions which affect
`the solubility of 'calcium-phosphate' would directly affect the
`nature ofthe precipitate complexes. To demonstrate this we tested
`live different calcium ctmccntrations between I25 and 250 mM
`
`in combination with It) dil'l'et'cnt phosphate concentrations
`between (HS and 6 rnM and determined the quantity of DNA
`remaining in solution after a centrifugation step (Fig. 5A). Willi
`acalcium concentration of 25“ ml“. the DNA was co-precipiurted
`fil a phosphate concentration of 0.5 mM or higher. When the
`calcium concentration was decreased. higher phosphate concentra-
`tions were needed to cit-precipitate DNA. Precipitatc-contples
`tonaation and binding of DNA could be initiated with each ot'thc
`calcium ct‘atccntratiorts used. yet at 12.5 mm calcium.
`the
`phosphate concentration had to be 2-1 ntM.
`To assess precipitate complex tormation directly. the mixture
`Isuspcnsiont was transferred into a spectrophotometer curettc.
`The non-precipitated solutions show no absorption at 320 nm
`”"2— 531. An increase of absorption at 320 nm indicates the
`appearance of a precipitate. which could be confirmed visually:
`“If: higher the phosphate concentration.
`the more cloudy the
`t'ttxpensron. Increasing concentrations of phosphate resulted for
`faith calcium—DNA mixture in atypical curve. showing ittcrea-‘iillg
`UPUCaI densities at higher phosphate concentrations. This assay is
`will reliable and gave reproducible results when performed with
`The same solutions repeatedly. It allows to distinguish pWCiPim'C-‘t
`WM With different phosphate concentrations together with the
`name calcium and DNA solutions.
`It should be noted.
`that
`alJStJIlJttttn at 320 nm is influenced by various parameters such as
`"i’St-tl “3’3. Particle number and structural characteristics of the
`PreF'PIFate complex and that an individual value does not give
`Ilttilrcations about the ratio of effects mediated by these different
`Pam-meters,
`tract-El?!” binding of DNA correlates with the appearance of a
`Wlpltate. Incomplete binding of DNA occurs in a very
`:imw concentration range of phosphate. While at a PhOFPha‘e
`mttit'ttrauon of 0.7? mM all DNA was associated thh the
`
`B
`
`SE
`
`._
`
`hi
`‘Fn0
`:3D
`o
`
`Figure 4. lilfecl of temperature on tol'ttiatlon of DNA—calcium phosphate
`anipitate crunpleses under a reduced phosphate concentration "run tnM.
`
`nntlrc other hand. all the DNA remained in the supernatant and
`almost no precipitate could be detected after 20 min. This
`indicates a reduced solubility of calcium phosphate at elevated
`temperature. and it appcw's that
`relatively small variations in
`temperature are able to afl'ect
`the kinetics of precipitate cotnples
`litrmation.
`
`Pfizer Ex. 1019
`Page 8 of 10
`
`

`

`600 NucleicAcidr Research. l996, Vol. 24. No. 4
`
`st
`
`3
`13
`c
`§
`9
`'0'D1
`
`A .
`
`B
`
`_. 250
`s
`g 200
`E
`?
`E
`4-
`o.
`T
`a
`"
`
`
`
`0
`
`0.1
`
`‘I
`mlvl of phosphate
`
`i U
`
`0
`0
`o
`3
`
`_|
`s
`£5
`7*
`ii"3
`‘9.
`i
`A
`E
`2
`
`titers
`normalizedstable
`
`
`— "- ‘GHT minus
`"“9... 30"” ”TX
`—"I— 100nM MTX
`
`1 .5
`1
`0.5
`0
`
`normatized transient titers
`
`2
`
`25
`
`_... — - y = 0232??) . 035'. is: in 0.62962
`
`I
`......... y = 0.10132 + 0.66160): Ft: 0.72945
`
`y = 0.20711 . 0.09am Fl: 0.621?
`|
`
`Figure 8. Correlation between transient and slahlc expression of [PA in 3 Sci of
`It] independent lmnslcclions. l-rotit each plate cells \vcrc seeded in selectite
`medium with increasing sclcctnc Pressure 1(ill'l'—. ill a.“ MTX and Ilium“
`MTXi 2 davs alter transt'cction. 'i‘ransicnt expression levels were assessed!
`days after translation stable expression levels were itsseswrl I'rtJIit pools ill
`cells after 2 weeks.
`
`the number of emerging clones alter the selection period oil—3
`weeks was found to be different ior each type of lranst'cction used.
`Most importantly. conditions that generated cliicicttt precipitate
`complexes for transient expression. also resulted in the largest
`number of stable clones (data not shown. 17).
`Figure 8 shows that expression levels generator] transiently (2 days
`after exposure of DNA to the cells) correlate to the protein levels
`observed in pools ol'stahie cells selected alter 2 weeks. Nonnuliretl
`values for transient expression were plotted against nonnaliaxl
`values representing stable expression of pools of recombinant cells
`selected under three different stringency conditions: medium tier
`ol‘glyeine. hypoxanthinc and tliyniidine tGHT—I. or GHT— media
`containing either 30 nM nicthotrexate 1MTX) or IOU nM MTX.
`Stable titers were Itorltialircd within each selection group. TM
`stringency ol' selcction. as expected. affected the stable lransfectiotl
`cliiciency (numbers of clones observed 1: higher selective prcfinm‘
`resulted in fewer clinics as well as in slightly smaller colony tilts
`Linear regression of the plotted data gave correlation coefficients
`of 0.83 for the values seen in the (.iHT— case [99% chance at
`correlation). 0.73 in the 30 nM MTX case [95% chance of
`correlation) and [3.62 in the Hit) nM MTX case (90% chancc 0i
`correlation). It is remarkable that the correlation for the till] 3““
`MTX ease remained good. in spite ol‘ the fact that the individual
`1900's ofstable clones consisted of only six clones (in the carcass
`WhiCh gave lowest transient titers) to 35 clones (in one ollht
`transfections which gave better transient titers). Therefore-837'}
`I
`assessment of a iransfection. using the supernatant just 2 days
`after DNA exposure. indicates whether few or many 300dClam l
`are likely to emerge weeks later.
`I
`
`1
`
`1
`
`i
`|
`
`.
`
`DISCUSSION
`
`The principle ofthe catcinm-phosphate—DNA t3re‘iii’ii‘ltioiI memo:
`originally developed by Graham and Van der Eh (I), *5 “ml
`Nevertheless the formation of an optimal DNA—calcium Dim--
`uflmfimmwisdmkth Eflmmw o8)hw““'-
`
`.
`
`‘
`
`l
`‘l
`
`i
`
`Pfizer EX. 1019 L
`
`Page 9 of 10
`
`Figure T. Asxessmcnt of various phosphate concentrations during the
`fom'iation of a precipitate (25 pg DNNIIii. 23 5C. IZS mM cailcruni.
`I. aim and
`pH 105}. iii! Quantity of soluble DNA in the supematant alter cenlnlugalion
`of the precipitation mix and optical density of the precipitation iiiixlurc at
`320 rim. I11] Transient expnrssion levels of tPA in the supe