High throughput genomic analysis:

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High throughput genomic analysis BEAMing
Dressman et al, 2003 Application to massively
parallel sequencing of genomes, transcriptomes,
SNP distinction)
2
BEAMing beads, amplification, emulsion,
magnetics cloning DNA moecules via PCR on
beads Each bead is provided with a single DNA
template and that seqeunce is amplified to yield
many copies of the complement immobilized on the
bead. (Dressman et al., Vogelstein lab. PNAS
2003. See reading assignment.)
All microspheres contain soluble primer, dNTPs,
Taq polymerase, etc.
No template or bead
Had one template
Had another template
No template
No bead
Or other ways to read out (e.g., CCD camera,
laser scanner for spot colors)
E.g., hybridization to a complementray probe can
distinguish 1 base differences at right temp.
3
Attached oligomers were pre-labeld red or green,
then mixed and emulsified. See single beads in
aqueous microspheres in oil.
oil
Aqueous microspheres (heterogeneous in size)
1 micron beads (here hybridized to 2 different
fluorescent probes)
4
FACS Fluorescence-activated cell sorter
Reports whether s droplet contain a cell or not.
In this paper, used to separate beads, not cells.
(Beads 1u size of an E. coli cell.)
Impart a charge on the recognized cell
Less than one cell or particle per droplet. Thus
the most that most droplets contain is one
particle.
Charged plates attract droplets containing a
particle of the opposite charge
Cells remain viable if treated with care.
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Histogram-type display
No fluorescence (background)
No. of cells
Red stained
Having this much fluorescence
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Scatter plot display
Analysis on 2 colors
One cell
Amount of green fluorescence (log)
You decide on the positions of of demarcations
Say, want high reds but low greens Instruct the
FACS to deflect cells in this quadrant. Collect
and grow or analyze further.
Amount of red fluorescence (log)
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A. Flow cytometry data 2-D plots where each
point represents one particle. Then contour lines
plotted around the point density. Here light
scattering (irrespective of wavelength) is
measured. Instrument can be set to reject data
from 2-bead doublets that scatter light more.
B-D. Amplified beads hybridized to 2 probes, one
specific to the S allele of a certain gene and
one specific to the L allele. The beads carry
the amplified PCR products corresponding to this
region from 3 human individuals. The blue
points come from micrspheres that contained both
tyes of PCR products from both alleles, despite
thehigh dilution.
Both signals
Red signal
Neither signal
Green signal
The L and S alleles can be distinguished by
probe hybridization. Here the DNAA region of
inerest was first amplified by an initial regular
PCR.
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Total genomic DNA can also be used as template
(A-B) if some of the forward primer is included
in solution as well as immobilized on the bead.
The initial PCR primers will have the appropriate
fixed end to allow the products to hybridize to
the primers on the beads.
C-D show that both alleles of the heterozygote
contribute equally to the cellular mRNA pool.
Beads that show no signal above background have
been eliminated from the display.
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Reconstruction experiimentshowing that the
ratio of beads of each color observed accurately
reflects the proportion input.
The PCR amplified DNA captured on a bead can be
sequenced. The data in B and C show that for each
bead a unique sequence is obtained.
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BEAMing applied to the early detection of colon
cancer A mutant DNA fragment that originated in
a mutant colon epithelial cell can be deteced in
the blood of an individual even when present as a
small minority.
Detection and quantification of mutations in the
plasma of patients with colorectal tumors.
Frank Diehl, Meng Li, Devin Dressma, Yiping He,
Dong Shen, Steve Szabo, Luis A. Diaz, Jr., Steven
N. Goodman, Kerstin A. David, Hartmut Juhl,
Kenneth W. Kinzler, and Bert Vogelstein
pp.1636816373 PNAS November 8, 2005 vol. 102
no. 45
Assay by primer extension (so no need to
distinguish plus or minus hybridization due to 1
base mismatch)
Note log scale
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BEAMing or polnioy formation appkied to high
throughput DNA sequencing Pyrosequencing and
sequencing by synthesis (SBS) Pyrosequencing
several views
APS adenosine phosphosulfate
Apyrase, a nucleotide degrading enzyme,
continuously degrades unincorporated dNTPs and
excess ATP. When degradation is complete, another
dNTP is added.
Biotage Web site and UCLA
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Pyrosequencing output
Limitation a long run of the same base cannot
be accurately read i.e., hard to tell the
difference between 10 As in a row and 11.
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Massively parallel DNA sequencing using
pyrosequencing
2. Denature and add to a PCR mix with one primer
immobilized on a nanobead and the other primer
soluble. 3. Mix with excess oil to form a
water-oil emulsion. All the reactants stay in the
aqueous microspheres that form. Template DNA is
sufficiently dilute such that each microshpere
contains 0 or 1 DNA template. 4. Run the PCR.
Anti-template strands will accumulate on the bead
as extensions to the immobilized primer. 5.
Recover the beads in aqueous solutin and let them
settle in nanowells. 6. Perform pyrosequencing
on the well array, noting light flash intensities
with a sanning sensor or CCD camera. 7. Computer
records flashes and yields 200,000 short
sequences from one array. 8. Available as a
commercial service (454, Inc). But one run
costs 12,000
Prepare DNA from a sample to be
analyzed.E.g., to sequence a genome, sample
could be from total DNA, with a constant PCR
primer ligated to genomic fragments. Or from a
cDNA sample, amplified by RT-PCR using a primers
with constant sequences included.
bead
Aqueous microsphere
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Sequencing by synthesis One way Start with a
very large number (500,000) of localized DNA
clones. Add a primer. Extend with a dTTP
labeled with a fluorescent dye and blocked on its
3 OH so that only one nucleotide can be added
(terminates) Wash. Take a digital picture of
all spots. Thise with an A as the first base
beyond the primer incorporate the dye and
fluorescne. Make a note of it position xxxx
red with fluorescent T. Add reagents to cleave
the dye moiety and the 3 )H block. Wash. Extend
with fluorescent dGTP blocked at its 3 OH. Etc.
etc. Probable limitation in read length
Incomplete cleavageof fluorescent moiety and/or
blocking agent. Solexa claims 25-50 base reads
of 1 million spots.
Design and Synthesis of a Photocleavable
Fluorescent Nucleotide 3'-O-Allyl-dGTP-PC-Bodipy-F
L-510 as a Reversible Terminator for DNA
Sequencing by Synthesis. Qinglin Meng, Dae Hyun
Kim, Xiaopeng Bai, Lanrong Bi,Nicholas J.
Turro,and Jingyue Ju Columbia University (not
Solexa) J. Org. Chem., 71 (8), 3248 -3252, 2006.
Two requirements 1) Localized amplification to
provide enough signal 2) Addition and removal of
a single template-directed base at a time,
Fluorescent group
3 OH blocking group
Colorless chain now ready for next base addition
Last base added to growing chain
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Solexa Web site (Solexa was just bought by
Illumina, 11/13/06) Animation
http//www.solexa.com/wt/page/tech_approach
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Hybridizes to a local pink immobilized primer
Hybridizes to a local blue immobilized primer
Sequencing
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Design and Synthesis of a Photocleavable
Fluorescent Nucleotide 3'-O-Allyl-dGTP-PC-Bodipy-F
L-510 as a Reversible Terminator for DNA
Sequencing by Synthesis. Qinglin Meng, Dae Hyun
Kim, Xiaopeng Bai, Lanrong Bi,Nicholas J.
Turro,and Jingyue Ju Columbia University J.
Org. Chem., 71 (8), 3248 -3252, 2006.
Fluorescent group
3 OH blocking group
Colorless chain now ready for next base addition
Last base added to growing chain
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Mammalian cell
genetics Introduction Genetics as a
subject (genetic processes that go on
in somatic cells
replicate, transmit, recombine, and express
genes) Genetics as a tool. Most
useful the less you know about a process. 4
manipulations of genetics 1- Mutation in
vivo (chnace selection, usually) targeted gene
knock-out or alteration in vitro site
directed or random cassette 2- Mapping
Organismic mating ?segregation, recombination
(e.g., transgenic mice) Cell culture cell
fusion segregation radiation hybrids FISH 3-
Gene juxtaposition (complementation)
Organisms matings ? heterozygotes Cell
culture cell fusion ? heterokaryons or hybrid
cells 4- Gene transfer transfection
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Mammalian cell genetics cont. Advantages of
cultured cells Numbers each cell is a genetic
individual, the ammmalian cell as a microorganism
Easily handle 10 million in an experiment, with
more effort 100 million. Homogeneity property
of the culture is more likely to b\represent the
property of each v\cell (compared to tissues)
Disadvantages of cultured mammalian cells
Limited phenotypes Limited differentiation in
culture (but some phenotypes available) No sex (
no regular segregation no easy way to
methodically re-assort alleles. Compare yeast
mating ? diploid, haploid strains
Mammalian cell lines Most genetic manipulations
use permanent lines, for the ability to do
multiple clonings. Primary, secondary cultures,
passages, senescence. Crisis, established cell
lines, immortality vs. unregulated growth. Most
permanent lines are immortalized, plus
"transformed, (plus have abnormal karyotypes)
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Mutation in cultured mammalian cells Problem of
epigenetic change Variants vs.
mutants Variants could include stable
heritable alterations in phenotype that are not
due to mutations i.e., heritable
switches in gene regulation (?, as in
differentiated tissues). DNA CpG methylation,
chromatn remodeling, histone acetylation/de-acetyl
ation, methylation, demethylation Diploidy as
in most somatic tissues, 2n. Haploidy as in
germ cells, 1n Aneuploidy any variation from
diploidy (e.g., trisomy 21) (2n1).
Heteroploidy More extreme variation in
chromosome number and more variation from cell to
cell. (e.g., 4n-20 sub-tetraploid)
The problem of diploidy and heteroploidy (compar
ed e.g., to yeast, or C. elegans, or Dros.) F2 ?
homozygotes not available) Recessive mutations
(most knock-outs) are masked.
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Solutions to diploidy problem Dominant
mutations only (too limited) Haploid cells
hasn't worked just modest chromosome reductions
in CHO Use haploid genes (XY) or functionally
haploid (XX, or allelic exclusion) too
restricitive Heterozygous loci (rare, despite
CHO reputation) Double mutants (incl. mutation
segregation, or mutation homozygosis via
mittotic recombination or loss reduplication
rare but can be done) (see next graphic) Heavy
mutagenesis, mutants/survivor increases but
mutants/ml decreases. How hard is it to get
mutants? What are the spontaneous and induced
mutation rates? Measurement of spontaneous
mutation rates. Rate vs. frequency (freq
includes accumulation). Spont
10-7/cell-generation Induced as high
as 10-3 /cell (EMS, UV) Note These same
considerations apply for the creation of double
knockout recessive tumor suppressor genes in
cancer.
EMS ethyl methane sulfonate UV ultraviolet
radiation)
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Loss of heterozygosity (LOH)by mitotic
recombination between homologous chromosomes
(rare, no synapsis as in meiosis))
or
-
-


-
-


-
-


Heterozygote
After homologous recombination (not sister
chromatid exchange)
1 homozygote 1 homozygote -
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Factors affectoing mutant isolation Mutagenesis.
Chemical and physical agents MNNG point
mutations (single base substitutions)
N-methyl-N-nitro-nitrosoguanidine EMS
Bleomycin
small deletions UV mostly point mutations but
also large deletions Ionizing radiation (X-rays,
gamma-rays) large deletions, rearrangements Dosa
ge kill 90 usually, as more killing leaves
too few survivors, including mutant survivors
Expression period dilute out WT molecules
(pre-existing protein and mRNA) Metabolic
cooperation WT toxic product can be transferred
cel to cell. Therefore somethimes necessary to
plate at low density. Dominant vs. recessive
mutations Dominants are rare (subtle change
e.g., drug resistant mutant enzyme), but
expression easily observed, Recessives easier to
get, (many ways to knock out a gene function) but
their expression is masked. Mutagen target
specificity (a particular base or base
combination (e.g., GG). Mutational spectra (hot
and cold spots are found). Strand specificity
transcribed strand is often preferentially
repaired.
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• Categories of cell mutants
• Exploitable metabolic pathways
• Purine and pyrimidine biosynthesis auxotrophs
• (auxotrophs require a nutrient in the medium that
  the WT doesnt)
• 1. Auxotrophs BUdR (BrdU) Kao and Puck. Kill
  growing cells. General method.
• Analogous to penicillin selection in
  prokaryotes.
• Many auxotrophs in amino acid or nucleotide
  biosynthetic pathways isolated
• 2. Drug resistance see sheet on nucleotide
  metabolism
• A. Mutant lacks toxifying enzyme
• e.g., HPRT (TGR), APRT (DAPR, 8-azaAR), TK
  (BrdUR)
• B. Enzyme target becomes a better discriminator
  
• (ouabain NaK ATPase pump
  alpha-amanitinRNA Pol II)
• C. Permeation changes influx blocked or efflux
  increased. (MDR via P-glycoprotein)
• D. Improved de-toxification via chelation,
  covalent modification,
• or overproduction of target (dhfr
  MTX-resistance via overproduction neoR
  neomycin phosphotransferase)

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• 3. Temperature-sensitive mutants cell cycle
  mutants.
• Tritiated amino acid suicide (aa-tRNA
  synthetases)
• 4. Antibodies. Lysis with complement. Targets
  cell surface constituents mostly (e.g., MHC)
• 5. Visual inspection at colony level
• A. Sib selection (G6PD)
• B. Replica plating (LDH)
• C. Secreted product (Iganti-Ig IP)
• FACS fluorescence-activated cell sorter.
• 1-D and 2-D fluorescence displays (cell
  surface Ag)
• Brute force
• (clonal biochemical analysis, e.g.,
  electrophoretic variants (e.g., Ig, isozymes)
• Direct genotype analysis (rare) (DNA isolation
  (via PCR and SSCP, single strand conformational
  polymorphism electrophoresis. Or DGGE denaturing
  gradient gel electrophoresis.
• MHC major histocompatability locus or proteins
  G6PD glucose-6-phosphate dehydrogenase LCH
  lactate dehydrogenase Ig immunoglobulin

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Cell fusion (for gene juxtaposition, mapping,
protein trafficking, ) Fusogenic agents PEG,
Sendai virus (syncytia promoting, as
HIV). Heterokaryons (2 nuclei), no cell
reproduction (limited times). (e.g., membrane
fluidity, nuclear shuttling, gene activation
(myoblasts) Hybrids (nuclei fuse, cells
reproduce). Small of heterokaryons. Complement
ation (e.g., auxotrophs with same
requirement) Dominance vs. recessiveness. Chromos
ome loss from hybrids ? Mapping chromosome
assignment. Synteny. Radiation hybrids linkage
analysis (sub-chromosomal regional assignments).
PEG polyethylene glycol, (1000 to 6000 MW)
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Cell fusion

Hprt, TK-
Parental cells
Hprt-, TK
HAT-
HAT-
PEG (polyethylene glycol, mw 6000 Sendai virus,
inactivated
Cell fusion
Heterokaryon (or, alternatively, homokaryon)
HAT medium
Hprt-, TK, Hprt TK-
HAT
Cell cycle, Nuclear fusion, Mitosis, survival
Hprt-, TK, Hprt TK-
membrane dynamics (lateral diffusion
Edidin), shuttling proteins (hnRNP A1
Dreyfuss), gene regulation (turn on myogenesis
Blau)
Hybrid cell
gene mapping (synteny Ruddle) gene regulation
(extinction of liver functions Weiss) Complement
ation (pyrimidine path Patterson)
Synteny genes physically linked on the same
chromosome are syntenic.
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Complementation analysis
Parental cells
Parental cells


gly-
gly-
gly-
gly-
Cell fusion
Cell fusion
glyA- glyA-
glyA- glyB-
Hybrid cell
Hybrid cell
Glycine-free Medium No growth, no
complementation, ?same gene (named glyA)
Glycine-free Medium Yes, growth, Yes,
complementation, ?different genes genes (named
glyA and glyB)
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Mapping genes to chromosomes
Hprt- x tk- ?Hybrid cell (Human x Rodent)
Reduced hybrid
Spontaneous chromosome loss (human
preferentially lost)
Hprt-, TK, Hprt TK-
Just passage and wait
Hprt-, TK, Hprt TK-
Correlate identified chromosome loss With loss of
phenotypic trait (isozyme, DNA sequence, etc.)
Isozymes enzyme variants that can be
distinguished from each other by physical
properties, often electrophoretic mobility in
native gels (net charge).
33
Ted Puck mutagenesis auxotrophic mutants in CHO
cells (U. Colo.)
Mary Weissturning off differentiation genes in
cell hybrids (Institut Pasteur)
Helen Blau Turning on muscle genes in
heterokaryons (Stanford)
Michael Edidin 2-D diffusion of proteins in the
cell membranein heterokaryons (Johns Hopkins)
Frank Ruddle Mapping by chromosome segregation
from cell hybrids.(Yale)
Nuclear-cytoplasmic shuttling in heterokaryons
(Penn)
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Transfection agents DEAE-dextran (toxic, OK for
transient) CaPO4 (co-precipitate) Electroporation
(naked DNA, high quick voltage ? transient
holes) Lipofection (multilamellar
liposomes) Polybrene (detergent?) Ballistic
(DNA-coated gold particles) Must traverse
cytoplasm. Much engulfed in lysosomes.
Inhibition of lysosomal function often helps
(chloroquin) Pechelosome 2000 KB
co-integration of high MW DNA. Separate plasmids
-gt same site (co-integration). Separate
transfections -gt separate locations Random or
semi-random (many) integration sites (unless
targeted) Low but real homologous recombination
rate History mammalian cell transfection
developed for practical use at Columbia (PS
Wigler Axel and Silverstein)
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Mike Wigler
Richard Axel
Saul Silverstein
History discovered for practical use at Columbia
(PS Wigler, Axel and Silverstein)
36
Transient transfection vs.
permanent cloned genes Unintegrated DNA
chromosomally
integrated Unnatural?
position effects
? Super-physiological expression (so
average many) levels (per transfected cell)
? Transient -gt 10-50 transfection efficiency
(stain) Permanents more like 0.001 per µg DNA
per cell (high). i.e., 106 cells ? 1000
colonies could be much less for certain types of
cells.
37
One the most dramatic first applications of gene
transfection from total DNA Transfer of the
growth-transformed phenotype ability to grow in
multilayers or in suspension in soft agar
(Weinberg, Wigler) DNA from tumor transfected
into growth-controlled mouse 3T3 cells. Look
for foci (one focus). Make a library from
growth-transformed transfectant. Screen for human
Alu repeat. Verify cloned DNA yields high
frequency of focus-forming transfectants. Isolate
cDNA by hybridization. Sequence. Identify gene
a dominant oncogene. Ras, a signaling protein
in a transducing pathway for sensing growth
factors
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Gene knockouts via homologous recombination.
ES cells and transgenic mice. Selection for
homologous recombinants via the loss of HSV TK
genes (Capecchi) tk homol. region YFG
homol. region tk (YFG your favorite
gene) tk homol. region YFG homol.
region tk Non-homologous recombination
favors ends tk is inserted, conferring
sensitivity to the drug gangcycliver (HSVtk
specific, not a substrate for human tk) Allele
replacements in cultured cell lines (e.g., APRT).
Most work in ES cells ? mice ? homozygosis via
F1 breeding Little work in cultured lines Myc
double sequenctial K.O. viable, sick (J.
Sedivy) Splicing factor (ASF) double K.O. in
chick DT40 lymphoid cells (high rate of
homologous recombination (J. Manley) Would be
lethal, but cover with inducible human ASF gene
(tet-off) Then add tet to analyze effects of gene
product removal
TK gene is removed during homologous
recombination. HSV Herpes simplex virustk
thymidine kinase
X
X
X
X
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neo
Double knockout of the ASF gene, a vital gene, by
homologous recombination
Chicken DT40 cells
One ASF gene allele disruted by homologous
recombination

hol
ASF-
hol
neo
neo
Tet-off promoter
pur
Hol histidinol resistance pur puromycin
resistance Drug resistance genes here chosen for
illustration.
Both alleles have been disrupted in some neoR,
holR cells
neo
ASF-
neo
tet
pur
ASF-
pur
X
Cell dies without ASF(follow events
biochemically)
cell viable(covered by human ASF gene
Wang, Takagaki, and Manley, Targeted disruption
of an essential vertebrate gene ASF/SF2 is
required for cell viability. Genes Dev. 1996 Oct
1510(20)2588-99.
40
Gene amplification Historically Methotrexate
resistance (Littlefield) High dihydrofolate
reductase (DHFR) enzyme activity, protein,
protein synthetic rate, translatable mRNA.
(Schimke) mRNA level, DNA level. Homogeneously
staining, expanded chromosomal regions (HSRs)
Biedler Nunberg HSR dhfr genes. Double
minute chromosomes. Amplicons. Big (300 KB). Can
shrink, migrate.
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Tritium grains from hybridized cDNA
42
Gene amplification
Homogeneously staining region FISH, here
43
Original locus?
HSR ? dmin upon DS break induced by a homing
endonuclease (I-SceI).
HSR homogeneously staining region Dmin double
minute chromosomes
Arnaud Coquelle, Lorène Rozier, Bernard
Dutrillaux and Michelle Debatisse ONCOGENE, 31
October 2002, Volume 21, Number 50, Pages
7671-7679 Induction of multiple double-strand
breaks within an hsr by meganucleaseI-SceI
expression or fragile site activation leads to
formation of double minutes and other chromosomal
rearrangements
44
Ampification models over-replication, unequal
sister chromatid exchange, breakage and fusion
(Tanaka paper). Map dhfr amplicons (Schimke,
Hamlin) 300 kb , but wide range Gene
amplification is rare in normal cells (Wahl,
Tslty). p53- allows. In nature rDNA in
oocytes, Drosophila chorion genes. In medicine
chemotherapy resistance (MDR, P-glycoprotein,
efflux pump) cancer (myc, ras) In
biotechnologyhigh level recombinant protein
production in mammalian cells
45
Fred Alt
Geoff Wahl
George Stark
46
Gene amplification for high level production in
CHO dhfr- cells.
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Reduction of folate to tetrahydrofolate
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Biosynthesis of glycine
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Biosynthesis of TMP
51
Biosynthesis of purine nucleotides
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DHFR- cells require G,H,T
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A different major system for high level Mab
production NS0 cells Mouse myeloma cells, high
IgG producers ? IgG variants NS0 No endogenous
IgG, but cell is a natural IgG secretor. Lack
glutamine synthetase (GS) glutamate NH3
ATP ? glutamine ADP Pi Vector MAb genes
driven by strong promoters (H-chain, L-chain)
GS cDNA gene (Bebbington) Select on
glutamine-free medium Inhibit GS with methionine
sulfoximine (gln analog) Select for GS
overproducers ---gt--gt (amplification of the GS
cDNA gene and linked Mab genes) Proprietary
(Lonza Biologics)
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Some other amplifiable genes
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Transfection strategies

• YFG (Your Favorite Gene) linked to a dhfr
  minigene on a single plasmid
• A. Insures co-integration
• B. Insures co-amplification
• YFG and dhfr on separate plasmids
• A. Allows a high ratio of YFG to dhfr to start

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Linked amp
CHO cells
57
Co-amp1
58
Co-amp3
(with or without pre-ligation)
59
kaufman
60
Co-amp2
61
Co-amp4
62
Amplification protocol