Cell, Vol. 16, 777-765,
`
`April 1979, Copyright
`
`0 1979 by MIT
`
`Transformation
`from Procaryotes
`
`of Mammalian Cells with Genes
`and Eucaryotes
`
`Sweet, Gek Kee Sim,
`Raymond
`Michael Wigler,*
`Barbara Weld, Angel Pellicer,
`Elizabeth
`Lacy,?
`Tom Maniatis,t
`Saul Silverstein
`and Richard Axel
`College
`of Physicians
`and Surgeons
`Columbia
`University
`New York, New York 10032
`
`Summary
`
`by
`
`cells with
`mammalian
`transformed
`We have stably
`and eucaryotic
`genes
`procaryotic
`precisely
`defined
`exist. The addition
`of
`for which no selective
`criteria
`a purified
`viral
`thymidine
`kinase
`(tk) gene
`to mouse
`cells
`lacking
`this enzyme
`results
`in the appearance
`of stable
`transformants
`which
`can be selected
`their
`ability
`to grow
`in HAT. These
`biochemical
`of
`transformants
`may
`represent
`a subpopulation
`other
`competent
`cells which
`are
`likely
`to
`integrate
`unlinked
`genes
`at frequencies
`higher
`than
`the gen-
`eral
`population.
`Co-transformation
`experiments
`were
`therefore
`performed
`with
`the viral
`tk gene and
`bacteriophage
`@X174,
`plasmid
`pBR322
`or
`the
`cloned
`chromosomal
`rabbit
`P-globin
`gene
`se-
`quences.
`Tk’
`transformants
`were
`cloned
`and ana-
`lyzed
`for co-transfer
`of additional
`DNA sequences
`by blot hybridization.
`In this manner, we have
`iden-
`tified mouse
`cell
`lines which
`contain multiple
`copies
`of CPX, pBR322
`and
`the
`rabbit
`/?-globin
`gene
`se-
`quences.
`The QX co-transformants
`were studied
`in
`greatest
`detail. The
`frequency
`of co-transformation
`is high: 15 of 16
`tk’
`transformants
`contain
`the QX
`sequences.
`Selective
`pressure was required
`to iden-
`tify co-transformants.
`From one
`to more
`than
`fifty
`@X sequences
`are
`integrated
`into high molecular
`weight
`nuclear
`DNA
`isolated
`from
`independent
`clones.
`Analysis
`of subclones
`demonstrates
`that
`the CPX genotype
`is stable
`‘through
`many
`genera-
`tions
`in
`culture.
`This
`co-transformation
`system
`should
`allow
`the
`introduction
`and stable
`integration
`of virtually
`any defined
`gene
`into
`cultured
`cells.
`Ligation
`to either
`viral
`vectors
`or selectable
`bio-
`chemical markers
`is not
`required.
`
`Introduction
`
`into cultured
`introduced
`genes can be stably
`Specific
`The rare
`trans-
`transfer.
`cells by DNA-mediated
`gene
`by biochemical
`selection.
`formant
`is usually detected
`isolated
`cells
`transformed
`In this manner, we have
`and viral genes
`coding
`for
`with a variety
`of cellular
`markers
`(Wigler
`et al., 1977,
`selectable
`biochemical
`1978,
`1979). The
`isolation
`of cells
`transformed
`with
`
`Cold Spring
`address:
`’ Present
`Harbor, New York 11724.
`t Division
`of Biology.
`California
`California
`91125.
`
`Harbor
`
`Laboratory,
`
`Cold Spring
`
`Institute
`
`of Technology,
`
`Pasadena,
`
`markers,
`for selectable
`do not code
`genes which
`however,
`is problematic
`since current
`transformation
`procedures
`are highly
`inefficient.
`This paper
`demon-
`strates
`the feasibility
`of co-transforming
`cells with
`two
`physically
`unlinked
`genes. Co-transformed
`cells can
`be
`identified
`and
`isolated when one of these genes
`codes
`for a selectable marker. We have used a viral
`thymidine
`kinase gene as a selectable marker
`to iso-
`late mouse cell
`lines which contain
`the
`tk gene along
`with either bacteriophage
`@Xl 74, plasmid
`pBR322
`or
`the cloned
`rabbit
`/I-globin
`gene
`sequences
`stably
`integrated
`into
`cellular
`DNA. The
`introduction
`of
`cloned eucaryotic
`genes
`into animal cells may provide
`a means
`for studying
`the functional
`consequences
`of
`DNA sequence
`organization.
`
`Results
`
`Design
`Experimental
`(tk) gene
`kinase
`thymidine
`of the purified
`The addition
`to mutant mouse cells
`lack-
`simplex
`virus
`from herpes
`in the appearance
`of stable
`transform-
`ing
`tk results
`the viral gene which can be selected
`ants expressing
`to row
`in HAT (Maitland
`and McDougall,
`by their ability
`et al., 1977).
`To obtain
`co-transform-
`1977; Wigler
`are exposed
`to
`the
`tk gene
`in
`the
`ants,
`cultures
`presence
`of a vast excess
`of a well defined
`DNA
`sequence
`for which hybridization
`probes
`are availa-
`ble. Tk’
`transformants
`are isolated
`and scored
`for the
`co-transfer
`of additional
`DNA sequences
`by molecular
`hybridization.
`
`of Mouse Cells with 8X1 74
`
`Co-transformation
`DNA
`exper-
`in co-transformation
`used QX DNA
`We initially
`the
`tk gene as the selectable marker.
`(PX
`iments with
`form DNA was cleaved with Pst
`I, which
`replicative
`a single site in the circular
`genome
`(Figure
`recognizes
`et al., 1977).
`500 pg of the purified
`tk gene
`1) (Sanger
`were mixed with
`l-l
`0 pg of Pst-cleaved @X replicative
`form DNA. This DNA was
`then added
`to mouse Ltk-
`cells using
`the
`transformation
`conditions
`previously
`in
`described
`(Wigler
`et al., 1979).
`After
`2 weeks
`selective medium
`(HAT),
`tk’
`transformants
`were ob-
`served at a frequency
`of one colony
`per 1 O6 cells per
`20 pg of purified
`gene. Clones were picked and grown
`into mass culture.
`also con-
`transformants
`tk’
`We then asked whether
`weight
`High molecular
`tained
`(PX DNA sequences.
`with
`the
`was cleaved
`DNA
`from
`the
`transformants
`restriction
`endonuclease
`Eco RI. which
`recognizes
`no
`sites
`in the @X genome.
`The DNA was fractionated
`by
`agarose
`gel electrophoresis
`and
`transferred
`to nitro-
`cellulose
`filters, and
`these
`filters were
`then annealed
`with nick-translated
`32P-QX DNA
`(blot hybridization)
`(Southern,
`1975;
`Botchan,
`Topp
`and Sambrook.
`1976; Pellicer
`et al., 1978). These annealing
`experi-
`
`Merck Ex. 1032, pg 1079
`
`

`
`Cell
`778
`
`8x 174
`
`Psi
`\
`
`ABCD
`
`E FGH
`
`I
`
`1.3
`
`0*7
`
`v
`
`.
`
`‘.’
`0.37
`
`I
`1.7
`
`3.7
`
`.‘.
`
`.
`
`2.0
`
`v
`
`,
`
`1. Cleavage Map of the @Xl 74 Genome
`Figure
`I (V), Hpa
`I, Hpa
`Pst
`Cleavage
`sites
`for
`the restriction
`endonucleases
`RFI and Pst
`I-linearized
`II (W and Hae
`Ill (I) are shown
`for circular
`@Xl 74 DNA
`(Sanger
`et al.. 1977). The numbers
`above
`the
`line refer
`to the sizes of the
`internal Hpa
`I fragments
`in kbp, while
`those below
`the line refer
`to the sizes of the Hpa
`II fragments.
`
`the seven
`that six of
`2) demonstrate
`(Figure
`ments
`bacteriophage
`se-
`had
`acquired
`transformants
`is not cut by
`the
`the
`(PX genome
`quences.
`Since
`enzyme Eco RI, the number
`of bands
`observed
`re-
`flects
`the minimum
`number
`of eucaryotic
`DNA
`frag-
`ments containing
`information
`homologous
`to (PX. The
`clones
`contain
`variable
`amounts
`of @X sequences.
`Clones @Xl and @X2 (Figure
`2, lanes A and C) reveal
`a single annealing
`fragment which
`is smaller
`than
`the
`OX genome.
`In these clones,
`therefore,
`only a portion
`of the
`transforming
`sequences
`persists.
`In lane D, we
`observe
`a tk’
`transformant
`(clone @X3) with no de-
`tectable CPX sequences.
`Clones @X4, 5,6 and 7 (lanes
`E, F, H and
`I) reveal numerous
`high molecular weight
`bands which are
`too closely
`spaced
`to count,
`indicat-
`ing
`that
`these
`clones
`contain multiple @X-specific
`fragments.
`These experiments
`demonstrate
`co-trans-
`formation
`of cultured mammalian
`cells with
`the viral
`tk
`gene and @X DNA.
`
`(PX
`
`to Identify
`
`Is Necessary
`Selection
`Transformants
`(PX DNA
`with
`transformation
`We next asked whether
`was restricted
`to the population
`of tk+ cells or whether
`a significant
`proportion
`of
`the original
`culture
`now
`contained
`0X sequences.
`Cultures were exposed
`to
`a mixture
`of the
`tk gene and @X DNA
`in a molar
`ratio
`of 1:2000
`or 1:20,000.
`Half of
`the cultures
`were
`plated under selective
`conditions,
`while
`the other half
`were plated
`in neutral media at low density
`to facilitate
`cloning. Both selected
`(tk+) and unselected
`(tk-) col-
`onies were
`picked,
`grown
`into mass
`culture
`and
`scored
`for
`the presence
`of
`(PX sequences.
`In
`this
`series of experiments,
`eight of the nine
`tk+ selected
`colonies
`contained
`phage
`information
`(Figure
`3). As
`
`with
`
`in Cells Transformed
`
`of CPX Sequences
`Identification
`Figure 2.
`@Xl 74 DNA and
`the HSV
`tk Gene
`tk
`the HSV
`with @Xl 74 DNA and
`Ltk-
`aprtt
`cells were
`transformed
`gene using
`salmon
`sperm DNA as carrier.
`Tk’
`transformants
`were
`selected
`by growth
`in HAT, cloned
`and grown
`into mass
`culture
`in
`HAT. High molecular
`weight DNA was extracted
`from
`seven
`inde-
`pendently
`isolated
`clones;
`15 pg of DNA
`from each were
`digested
`with Eco RI, electrophoresed
`through
`1% agarose
`gels, denatured
`in
`situ and
`transferred
`to nitrocellulose
`filters which were
`then annealed
`with
`‘*P-@X174
`DNA
`(5 x 10’ cpm/gg)
`to identify
`co-transformants.
`Lanes B and G are Eco RI digests
`of 32P-adenovirus
`2 DNA;
`the six
`bands are 20.3, 4.2, 3.6, 2.6, 2.2 and 1.6 kbp. Lanes A, C. D. E. F.
`H and
`I are
`the seven
`independently
`isolated
`clones @Xl -7,
`respec-
`tively. Only clone OX3
`(lane D) lacks
`detectable @X sequences.
`
`contained
`the clones
`experiments,
`the previous
`in
`of
`none
`In contrast,
`of
`(PX DNA.
`varying
`amounts
`from neutral medium
`fifteen clones picked
`at random
`contained
`any (PX information
`(data not shown). Thus
`the addition
`of a selectable
`marker
`facilitates
`the
`identification
`of those cells which contain OX DNA.
`
`DNA
`into Cellular
`Integrated
`Are
`QX Sequences
`Cleavage
`of DNA
`from
`(PX transformants
`with Eco RI
`(Figure
`2) generates
`a series of fragments which con-
`tain (PX DNA sequences.
`These
`fragments may reflect
`multiple
`integration
`events. Alternatively,
`these
`frag-
`ments could
`result
`from
`tandem arrays of complete
`or
`partial @X sequences
`which
`are not
`integrated
`into
`cellular DNA. To distinguish
`between
`these possibili-
`ties,
`transformed
`cell DNA was cut with Barn HI or Eco
`RI, neither of which cleaves
`the (PX genome.
`If the @X
`DNA sequences
`were not
`integrated,
`neither of these
`enzymes would
`cleave
`the CPX fragments.
`Identical
`patterns would
`be generated
`from undigested
`DNA
`
`Merck Ex. 1032, pg 1080
`
`

`
`Transformation
`779
`
`of Mammalian
`
`Cells
`
`ABCDEFGHI
`
`JK
`
`ABC
`
`DE
`
`FGH
`
`I.1
`
`in tk+ Transformants
`3. QX Sequences
`Figure
`2,
`to Figure
`legend
`as described
`in the
`Cells were
`co-transformed
`the other
`half were
`were
`fed HAT, while
`and half of
`the cultures
`conditions
`in DME. Nine colonies
`were
`se-
`replated
`under
`cloning
`lected
`in HAT and assayed
`for @X sequences
`as described
`(see
`Figure
`2). Lanes A and K each contain
`30 pg (2 gene equivalents)
`of
`Pst l-linearized @Xl 74 DNA. Lanes B-J contain
`Eco RI-digested
`DNA
`from nine
`independently
`isolated
`tk’
`transformants.
`Only one clone
`(lane E) does
`not contain @X sequences.
`None
`of
`fifteen
`clones
`isolated without
`selection
`contained
`QX sequences
`(blot not shown).
`
`from DNA cleaved with either of these enzymes.
`and
`If the sequences
`are
`integrated,
`then Barn HI and Eco
`RI should
`recognize
`different
`sites
`in
`the
`flanking
`cellular DNA and generate
`unique
`restriction
`patterns.
`DNA from clones OX4 and @X5 was cleaved with Barn
`HI or Eco RI and analyzed
`by Southern
`hybridization
`(Figure
`4: clone
`4,
`lanes D and E; clone
`5,
`lanes F
`and G).
`In each
`instance,
`the annealing
`pattern with
`Eco RI fragments
`differed
`from
`that observed
`with
`the
`Barn HI fragments.
`Furthermore,
`the profile
`obtained
`with undigested
`DNA
`reveals
`annealing
`only
`in very
`high molecular
`weight
`regions with no discrete
`frag-
`ments
`observed
`(data
`not shown).
`Similar
`observa-
`tions were made on clone @Xl
`(data not shown). Thus
`most of the QX sequences
`in these
`three
`clones are
`integrated
`into cellular DNA.
`
`of the 8X Sequences
`Localization
`Intracellular
`cells
`of (PX sequences
`in
`transformed
`The
`location
`Nuclear
`by subcellular
`fractionation.
`was determined
`the @X
`and
`fractions were prepared,
`and cytoplasmic
`by blot
`content
`of each was assayed
`DNA sequence
`that 95%
`The data
`(not shown)
`indicate
`hybridization.
`of the QX sequences
`are
`located
`in the nucleus. High
`and
`low molecular
`weight nuclear DNA was prepared
`by Hirt
`fractionation
`(Hirt,
`1967). Hybridization
`with
`
`in bX Co-transform-
`
`of Sequence
`
`Representation
`
`Figure 4. Extent
`ants
`clones OX4 and
`co-transformant
`from
`weight DNA
`High molecular
`cPX5 was digested
`with either Eco RI. Barn HI, Hpa
`I or Hpa
`II and
`analyzed
`for
`the presence
`of QX sequences
`as described
`in
`the
`legend
`to Figure
`2. (Lanes
`B and
`I) 50 pg (4 gene equivalents)
`of QX
`RFI DNA digested
`with Hpa
`I and Hpa
`II. respectively.
`(Lanes A, D, E
`and H) 15 pg of clone @X4 DNA digested
`with Hpa
`I, Eco RI. Barn HI
`and Hpa
`II, respectively,
`and analyzed
`for @X sequences
`by blot
`hybridization.
`(Lanes C. F. G and J) 15 rig of clone @X5 DNA digested
`with Hpa
`I, Eco RI. Barn HI or Hpa
`II, respectively.
`
`than
`that more
`indicates
`two fractions
`these
`DNA from
`with
`the high
`co-purifies
`information
`95% of the OX
`molecular
`weight DNA
`fraction.
`The small amount
`of
`hybridization
`observed
`in the supernatant
`fraction
`re-
`veals a profile
`identical
`to that of the high molecular
`weight DNA, suggesting
`contamination
`of this fraction
`with high molecular
`weight DNA.
`
`of the @X
`
`of Sequence
`
`Representation
`
`Extent
`Genome
`transformed
`from
`of DNA
`profiles
`The annealing
`clones
`digested with enzymes
`that do not cleave
`the
`(PX genome
`provide
`evidence
`that
`integration
`of CPX
`sequences
`has occurred
`and allow us to estimate
`the
`number
`of @X sequences
`integrated.
`Annealing
`pro-
`files of DNA
`from
`transformed
`clones
`digested
`with
`enzymes which cleave within
`the @X genome
`allow us
`to determine what proportion
`of the genome
`is present
`and how
`these sequences
`are arranged
`following
`in-
`tegration.
`Cleavage
`of @X with
`the enzyme
`Hpa
`I
`generates
`three
`fragments
`for each
`integration
`event
`(see Figure
`1):
`two
`“internal”
`fragments
`of 3.7 and
`1.3 kb which
`together
`comprise
`90% of the
`(PX ge-
`nome,
`and one
`“bridge”
`fragment
`of 0.5 kb which
`spans
`the Pst
`I cleavage
`site. The annealing
`profile
`observed
`when clone @X4
`is digested
`with Hpa
`I is
`shown
`in Figure
`4,
`lane A. Two
`intense
`bands
`are
`oberved
`at 3.7 and 1.3 kb. A less
`intense
`series of
`
`Merck Ex. 1032, pg 1081
`
`

`
`Cell
`780
`
`is also observed,
`weight
`of higher molecular
`bands
`represent @X sequences
`of which
`probably
`some
`adjacent
`to cellular DNA. These
`results
`indicate
`that
`at
`least 90% of
`the @X genome
`is present
`in these
`I
`cells.
`It is worth noting
`that
`the
`internal
`1.3 kb Hpa
`fragment
`is bounded
`by an Hpa
`I site only 30 bp from
`the Pst I cleavage
`site. Comparison
`of the
`intensities
`of the
`internal
`bands with known
`quantities
`of Hpa-I-
`cleaved
`(PX DNA suggests
`that
`this clone
`contains
`approximately
`100 copies of the @X genome
`(Figure
`4,
`lanes A and B). The annealing
`patten
`of clone 5
`DNA cleaved with Hpa
`I is more complex
`(Figure
`4,
`lane C).
`If internal
`fragments
`are present,
`they are
`markedly
`reduced
`in intensity;
`instead, multiple bands
`of varying molecular weight are observed.
`The 0.5 kb
`Hpa
`I fragment which bridges
`the Pst I cleavage
`site
`is not observed
`for either
`clone @X 4 or clone @X5
`(data not shown).
`of clone @X4 and @X5 DNA was
`A similar analysis
`performed
`with
`the enzyme
`Hpa
`II. This enzyme
`cleaves
`the @X genome
`five
`times,
`thus generating
`four
`“internal”
`fragments
`of 1.7, 0.5, 0.5 and 0.2 kb,
`and a 2.6 kb “bridge”
`fragment which
`spans
`the Pst
`I cleavage
`site (Figure
`1 ):The
`annealing
`patterns
`for
`Hpa
`II-cleaved DNA from @X clones 4 and 5 are shown
`in Figure 4 (clone @X4,
`lane H; clone @X5,
`lane J). In
`each clone an intense
`1.7 kb band
`is observed,
`con-
`sistent with
`the
`retention
`of at least
`two
`internal Hpa
`II sites. The 0.5 kb
`internal
`fragments
`can also be
`observed,
`but
`they are not shown on
`this gel. Many
`additional
`fragments,
`mostly
`of higher molecular
`weight, are also present
`in each clone. These presum-
`ably
`reflect
`the multiple
`integration
`sites of @X DNA in
`the cellular
`genome. The 2.6 kb fragment
`bridging
`the
`Pst I cleavage
`site, however,
`is absent
`from clone @X4
`(Figure
`4,
`lane H). Reduced
`amounts
`of annealing
`fragments
`which
`co-migrate
`with
`the 2.6 kb Hpa
`bridge
`fragment
`are observed
`in clone
`(PX 5 (Figure
`4, lane J). Similar
`observations
`were made
`in experi-
`ments with
`the enzyme Hae
`Ill. The annealing
`pattern
`of Hae Ill-digested
`DNA from
`these clones
`is shown
`in
`Figure
`5 (clone @X4,
`lane B; clone @X5,
`lane C).
`In
`accord with our previous
`data,
`the 0.87
`kb Hae
`III
`bridge
`fragment
`spanning
`the Pst site
`is absent
`or
`present
`in reduced
`amount
`in transformed
`cell DNA.
`Thus
`in general
`“internal”
`fragments
`of @X are
`found
`in
`these
`transformants,
`while
`“bridge”
`fragments
`which
`span
`the Pst
`I cleavage
`site are
`reduced
`or
`absent
`(see Discussion).
`
`II
`
`Genotype
`of the Transformed
`Stability
`of selecta-
`on the
`transfer
`Our previous
`observations
`indicate
`that the transformed
`ble biochemical
`markers
`for hundreds
`of generations
`phenotype
`remains
`stable
`If
`under
`selective
`pressure.
`if cells are maintained
`maintained
`in neutral medium,
`the
`transformed
`phe-
`notype
`is lost at frequencies
`which
`range
`from co.1
`to as high as 30% per generation
`(Wigler et al., 1977,
`
`ABCD
`
`”
`
`.*:
`
`,a/
`
`I
`
`+
`
`Pattern
`
`of DNA
`
`from Hae
`
`Ill-Digested
`
`@X Co-
`
`5. Annealing
`Figure
`transformants
`from clones @X4 and OX5 was digested
`weight DNA
`High molecular
`the annealing
`profile was compared
`with
`that of Hae
`with Hae
`Ill. and
`Ill-digested
`BXRFI DNA. Lanes A and D contain
`30 and 50 pg (2 and
`4 gene equivalents,
`respectively)
`of Hae
`Ill-digested
`OXRFI DNA.
`Lanes B and C contain
`15 pg of Hae
`Ill-digested
`DNA
`from
`clones
`OX4 and 0X5.
`respectively.
`The sizes
`of the prominent
`8X Hae
`Ill
`fragments
`in lanes A and D are 1350.1080.870
`and 600 base pairs.
`
`the expres-
`to study
`1979). The use of transformation
`upon
`the stability
`of the
`sion of foreign genes depends
`is an important
`consider-
`transformed
`genotype.
`This
`no selctive
`criteria
`are
`ation with genes
`for which
`available. We assume
`that the presence
`of (PX DNA
`in
`our
`transformants
`confers
`no selective
`advantage
`on
`the recipient
`cell. We therefore
`examined
`the stability
`of the CPX genotype
`in the descendants
`of two clones
`after numerous
`generations
`in culture.
`Clones @X4
`and @X5, both containing multiple
`copies of @X DNA,
`were subcloned
`and six independent
`subclones
`from
`each original
`clone were picked and grown
`into mass
`culture. DNA
`from each of these subclones
`was
`then
`digested with either Eco RI or Hpa
`I, and
`the annealing
`profiles
`of @X-containing
`fragments
`were
`compared
`with those of the original
`parental
`clone. The annealing
`pattern
`observed
`for four of the six @X4 subclones
`virtually
`identical
`to that of the parent
`(Figure
`6A).
`
`is
`In
`
`Merck Ex. 1032, pg 1082
`
`

`
`Transformation
`701
`
`of Mammalian
`
`Cells
`
`a
`
`ABCDEFGHIJ
`
`without
`
`significant
`
`loss or translocation
`
`of information.
`
`in
`
`into Mouse Cells
`DNA
`of pBR322
`Integration
`We have extended
`our observations
`on co-transfor-
`mation
`to the EK2-approved
`bacterial
`vector, plasmid
`pBR322.
`pBR322
`linearized
`with Barn HI was mixed
`with
`the purified
`viral
`tk gene
`in a molar
`ratio of 1000:
`1. Tk’
`transformants
`were selected
`and scored
`for the
`presence
`of pBR322
`sequences.
`The Bgl
`I restriction
`map of Barn HI linearized
`pBR322
`DNA
`is shown
`Figure
`7. Cleavage
`of this DNA with Bgl
`I generates
`two
`internal
`fragments
`of 2.4 and 0.3 kb. The se-
`quence
`content
`of
`the pBR322
`transformants
`was
`determined
`by digestion
`of transformed
`cell DNA with
`Bgl
`I followed
`by annealing
`with 3’P-labeled
`plasmid
`DNA. Four of
`five clones
`screened
`contained
`pl3R
`sequences.
`Two of these clones
`contained
`the 2.4 kb
`kb
`internal
`fragment
`(Figure
`8). The 0.3
`fragment
`would not be detected
`on these gels. From
`the inten-
`sity of the 2.4 kb band
`in comparison
`with controls,
`we conclude
`that multiple
`copies of this
`fragment
`are
`present
`in these
`transformants.
`Other bands are ob-
`served which
`presumably
`represent
`the segments
`of
`pBR322
`attached
`to cellular DNA.
`
`of Mouse Cells with
`
`the Rabbit
`
`fl-
`
`Transformation
`Globin Gene
`genes may
`eucaryotic
`with purified
`Transformation
`of cloned
`the expression
`for studying
`provide
`a means
`therefore
`host. We have
`genes
`in a heterologous
`with
`the
`experiments
`performed
`co-transformation
`rabbit p major globin
`gene which was
`isolated
`from a
`cloned
`library
`of rabbit
`chromosomal
`DNA
`(Maniatis
`et al., 1978). One
`/I-globin
`clone designated
`RPG-1
`(Lacy et al., 1978)
`consists
`of a 15 kb
`rabbit DNA
`fragment
`carried
`on the bacteriophage
`h cloning
`vec-
`tor Charon
`4a.
`Intact DNA
`from
`this clone
`(RPG-1)
`was mixed with
`the viral
`tk DNA at a molar
`ratio of
`1OO:l
`, and
`tk’
`transformants
`were
`isolated
`and ex-
`amined
`for
`the presence
`of rabbit globin
`sequences.
`9.
`A restriction
`map of R/3G-1
`is shown
`in Figure
`Cleavage
`of RPG-1 with
`the enzyme Kpn
`I generates
`a 4.7 kb
`fragment
`which
`contains
`the entire
`rabbit
`P-globin
`gene.
`This
`fragment
`was purified
`by gel
`electrophoresis
`and
`nick-translated
`to generate
`a
`probe
`for subsequent
`annealing
`experiments.
`The p-
`globin genes of mouse and
`rabbit are partially
`homol-
`ogous,
`although
`we do not observe
`annealing
`of the
`rabbit P-globin
`probe with Kpn-cleaved
`mouse DNA
`under
`our experimental
`conditions
`(Figure
`10,
`lanes
`C, D and G).
`In contrast,
`cleavage
`of rabbit
`liver DNA
`with Kpn
`I generates
`the expected
`4.7 kb globin band
`(Figure
`10, lane B). Cleavage
`of transformed
`cell DNA
`with
`the enzyme Kpn
`I generates
`a 4.7 kb
`fragment
`containing
`globin-specific
`information
`in six of
`the
`eight
`tk’
`transformants
`examined
`(Figure
`10).
`In two
`of the clones
`(Figure
`10,
`lanes E and H), additional
`rabbit globin
`bands are observed
`which probably
`re-
`
`b
`
`of Co-transformants
`in Subclones
`6. @X Sequences
`Figure
`(a) Annealing
`profiles
`of DNA
`from parental
`clone @X4 digested with
`Eco RI (lane A) and Hpa
`I (lane H) are compared
`with DNA
`from six
`independent
`subclones
`digested
`with either
`Eco RI (lanes B-G)
`or
`Hpa
`I (lanes
`I-N).
`of clone @X5 was
`four subclones
`from
`weight DNA
`(b) High molecular
`Eco RI or Hpa
`II and compared
`with
`with either
`isolated,
`cleaved
`parental
`clone @X5 DNA. Lanes A and F contain
`clone @X5 DNA
`digested
`with Eco RI and Hpa
`II, respectively.
`DNA
`from
`four
`inde-
`pendently
`isolated
`subclones
`digested
`with either Eco RI (lanes B-E)
`or Hpa
`II (lanes G-J) was analyzed
`by blot hybridization.
`
`ap-
`fragment
`Eco RI
`an additional
`two subclones,
`is of identical molecular
`weight
`in both.
`peared which
`resulted
`from genotypic
`heterogeneity
`This may have
`clone prior
`to subcloning.
`The patterns
`in the parental
`the subclones
`of @X5 are again virtually
`obtained
`for
`identical
`to the parental
`annealing
`profile
`(Figure
`6B).
`These data
`indicate
`that @X DNA
`is maintained
`within
`the ten subclones
`examined
`for numerous
`generations
`
`Merck Ex. 1032, pg 1083
`
`

`
`Cell
`782
`
`PBR 322
`
`0.3
`
`,
`
`2.4
`
`v
`v
`7. Cleavage Map of pBR322
`Figure
`The Bgl I restriction
`endonuclease
`map
`DNA
`is shown.
`The
`fragment
`sizes
`are
`Sutcliffe
`(personal
`communication).
`
`A B
`
`c
`
`D
`
`v
`
`I,3
`
`4
`
`for Barn HI-linearized
`in kbp, as determined
`
`pBR322
`by G.
`
`sites
`the Kpn
`least one of
`loss of at
`the
`from
`sult
`globin
`rabbit
`during
`transformation.
`The number
`of
`is variable.
`In
`genes
`integrated
`in these
`transformants
`10, lanes A and
`comparison
`with control
`lanes
`(Figure
`L), some clones
`contain
`a single
`copy of
`the gene
`(lanes
`I, J and K), while others contain multiple
`copies
`of
`this heterologous
`gene
`(lanes E, F and H). These
`results demonstrate
`that cloned eucaryotic
`genes can
`be
`introduced
`into cultured mammalian
`cells by co-
`transformation.
`
`Competence
`
`Is Not Stably
`
`Transformation
`Inherited
`of
`of a subpopulation
`the existence
`Our data suggest
`cell
`transformation-competent
`cells within
`the
`total
`trait,
`population.
`If competence
`is a stably
`inherited
`then cells selected
`for transformation
`should be better
`recipients
`in subsequent
`gene
`transfer
`experiments
`than
`their parental
`cells. Two
`results
`indicate
`that as
`in procaryotes,
`competence
`is not stably heritable.
`In
`the
`first series of experiments,
`a double mutant, Ltk-
`aprt-
`(deficient
`in both
`tk and aprt), was
`transformed
`to either
`the tk’ aprt- or the tk- aprt’ phenotype
`using
`cellular
`DNA as donor
`(Wigler
`et al., 1978,
`1979).
`These clones were
`then
`transformed
`to the
`tk’ aprt’
`phenotype.
`The
`frequency
`of the second
`transforma-
`In
`tion was not significantly
`higher
`than
`the
`first.
`another
`series of experiments,
`clones @X4 and @X5
`were used as recipients
`for
`the
`transfer
`of a mutant
`folate
`reductase
`gene which
`renders
`recipient
`cells
`resistant
`to methotrexate
`(mtx). The cell
`line A29
`MtxR”’ contains
`a mutation
`in the structural
`gene
`for
`dihydrofolate
`reductase,
`reducing
`the affinity
`of this
`enzyme
`for methotrexate
`(Flintoff, Davidson
`and Sim-
`inovitch,
`1976). Genomic DNA from
`this line was used
`to transform
`clones
`(PX4 and @X5 and Ltk- cells. The
`frequency
`of transformation
`to mtx resistance
`for
`the
`to
`QX clones was
`identical
`that observed
`with
`the
`parental
`Ltk- cells. We conclude
`that competence
`is
`not a stably heritable
`trait and may
`therefore
`be a
`transient
`property
`of cells.
`
`Discussion
`
`mam-
`transformed
`studies, we have stably
`In these
`and
`procaryotic
`cells with precisely
`defined
`malian
`exist.
`eucaryotic
`genes
`for which no selective
`criteria
`Our chosen experimental
`design derives
`from studies
`of
`transformation
`in bacteria
`which
`indicate
`that a
`
`2.4
`.-
`1.6
`
`in Co-transformants
`Sequences
`Figure 8. Physical Map of pBR322
`and
`the
`viral
`tk gene
`DNA and
`Cells were
`exposed
`to pBR322
`from
`three
`independent
`weight DNA
`selected
`in HAT. High molecular
`clones was digested with Bgl I and electrophoresed
`on a 1% agarose
`gel. The DNA was denatured
`in situ and
`transferred
`to nitrocellulose
`filters which were annealed with 32P-pBR322
`DNA.
`(Lane A) 5 pg of
`pBR322
`DNA digested
`with Bgl
`I; (lanes
`B-D)
`15 pg of DNA
`from
`three
`independent
`tk+
`transformants.
`
`is compe-
`of cells
`subpopulation
`small but selectable
`Hotchkiss,
`1955;
`(Thomas,
`tent
`in
`transformation
`1959; Tomasz and Hotchkiss,
`1964; Spizizen.
`Reilly
`and Evans, 1966).
`If this
`is also
`true
`for animal
`cells,
`then biochemical
`transformants
`will
`represent
`a sub-
`population
`of competent
`cells which are
`likely
`to
`in-
`tegrate
`other
`unlinked
`genes
`at
`frequencies
`higher
`than
`the general
`population.
`Thus,
`to
`identify
`trans-
`formants
`containing
`genes which provide
`no selecta-
`ble
`trait, cultures were co-transformed
`with a physi-
`cally
`unlinked
`gene which
`provided
`a selectable
`marker.
`This co-transformation
`system
`should
`allow
`the
`introduction
`and stable
`integration
`of virtually
`any
`defined
`gene
`into cultured
`cells. Ligation
`to either viral
`vectors
`or selectable
`biochemical
`markers
`is not
`re-
`quired.
`
`Merck Ex. 1032, pg 1084
`
`

`
`Transformahon
`783
`
`of Mammalian
`
`Cells
`
`RSG-1
`
`16.5
`
`4.7
`
`v
`
`v
`
`Phage Clone RPG-1
`9. Physical Map of Rabbit P-Globin
`Figure
`Cleavage
`sites
`for
`the
`restriction
`endonuclease
`Kpn
`I within R/3G-1
`are shown
`(Lacy
`et al., 1979). The numbers
`to the sizes of the
`fragments
`in kbp.
`
`refer
`
`us-
`were performed
`experiments
`Co-transformation
`as the selectable
`biochemical
`ing
`the HSV
`tk gene
`of this purified
`tk gene
`to mouse
`marker. The addition
`cells
`lacking
`thymidine
`kinase
`results
`in the appear-
`ance of stable
`transformants
`which
`can be selected
`by their ability
`to grow
`in HAT. Tk’
`transformants
`were
`cloned and analyzed
`by blot hybridization
`for co-trans-
`fer of additional
`DNA sequences.
`In this manner, we
`have constructed
`mouse cell
`lines which contain mul-
`tiple copies of @X, pBR322
`and
`rabbit
`/I-globin
`gene
`sequences.
`result
`could
`these observations
`that
`The suggestion
`procaryotic
`cells
`in our cultures
`is
`from contaminating
`At least one of the rabbit P-globin
`highly
`improbable.
`expresses
`polyadenylated
`rab-
`mouse
`transformants
`bit /?-globin
`RNA sequences
`as a discrete
`9s cyto-
`plasmic
`species
`(B. Wold et al., manuscript
`in prepa-
`ration). The elaborate
`processing
`events
`required
`to
`generate
`9s globin RNA correctly
`are unlikely
`to occur
`in procaryotes.
`in greatest
`were studied
`The
`(PX co-transformants
`is high: 14
`detail. The
`frequency
`of co-transformation
`of 16
`tk’
`transformants
`contain @X sequences.
`The
`(PX sequences
`are
`integrated
`into
`high molecular
`weight nuclear DNA. The number
`of integration
`events
`varies
`from one
`to more
`than
`fifty
`in
`independent
`clones. The extent of the bacteriophage
`genome
`pres-
`ent within a given
`transformant
`is also variable; while
`some clones
`have
`lost up
`to half
`the genome,
`other
`clones
`contain
`over 90% of the @X sequences.
`Anal-
`ysis of subclones
`demonstrates
`that the (PX genotype
`is stable
`through many generations
`in culture. Similar
`conclusions
`are emerging
`from
`the characterization
`of
`the pBR322
`and globin
`gene co-transformants.
`Hybridization
`analysis
`of restriction
`endonuclease-
`cleaved
`transformed
`cell DNA allows us to make some
`preliminary
`statements
`on the nature of the
`integration
`intermediate.
`Only
`two (PX clones have been examined
`in detail.
`In both clones,
`the donor DNA was Pst
`linearized
`(PX DNA. We have attempted
`to distinguish
`between
`the
`integration
`of a linear or circular
`inter-
`mediate.
`If either precise
`circularization
`or the forma-
`tion of
`linear
`concatamers
`had occurred
`at the Pst
`cleavage
`site, and
`if integration
`occurred
`at random
`points
`along
`this DNA, we would
`expect
`cleavage
`maps of transformed
`cell DNA
`to mirror
`the circular
`@X map. The bridge
`fragment,
`however,
`is not ob-
`served or is present
`in reduced
`amounts
`in digests of
`
`I-
`
`I
`
`in Mouse DNA
`Is Present
`Gene
`10. The Rabbit P-Globin
`Figure
`tk gene and selected
`the viral
`Cells were exposed
`to RPG-1 DNA and
`independent
`clones
`in HAT. High molecular
`weight DNA
`from eight
`was digested
`with Kpn
`I and electrophoresed
`on a 1% agarose
`gel.
`The DNA was
`denatured
`in situ and
`transferred
`to nitrocellulose
`filters, which were
`then annealed with a 32P-labeled
`4.7 kbp
`fragment
`containing
`the rabbit b-globin
`gene.
`(Lanes A and L) 50 pg of the 4.7
`kbp Kpn
`fragment
`of R/3G-1;
`(lane B) 15 pg of
`rabbit
`liver DNA
`digested
`with Kpn;
`(lane C) 15 pg of Ltk-
`aprt- DNA;
`(lanes D-K)
`15
`pg of DNA
`from each of eight
`independently
`isolated
`tk’
`transform-
`ants.
`
`restriction
`three different
`cell DNA with
`transformed
`are
`in ac-
`The
`fragments
`observed
`endonucleases.
`cord with a model
`in which CPX DNA
`integrates
`as a
`linear molecule.
`Alternatively,
`it is possible
`that
`intra-
`molecular
`recombination
`of CPX DNA occurs,
`resulting
`in circularization
`with deletions
`at the Pst termini
`(Lai
`and Nathans,
`1974). Random
`integration
`of this cir-
`cular molecule would generate
`a restriction map sim-
`ilar
`to that observed
`for clones @X4 and @X5. Other
`more
`complex models
`of events
`occurring
`before,
`during
`or after
`integration
`can also be considered.
`Although
`variable
`amounts
`of DNA may be deleted
`from
`termini
`during
`transformation,
`most copies
`of
`integrated @X sequences
`in clone @X4 retain
`the Hpa
`I site, which
`is only 30 bp from
`the Pst I cleavage
`site.
`Whatever
`the mode of integration,
`it appears
`that cells
`can be stably
`transformed
`with
`long stretches
`of donor
`DNA. We have observed
`transformants
`containing
`contiguous
`stretches
`of donor DNA 50 kb
`long
`(B.
`Wold et al., unpublished
`studies).
`transformed
`cells
`We have attempted
`to
`identify
`of selective
`pres-
`with @X sequences
`in the absence
`sure. Cultures were exposed
`to CPX and
`tk DNA and
`c