Hprt, TK ,

1
Mapping genes to chromosomes
Hybrid cell
Reduced hybrid
Spontaneous chromosome loss (human
preferentially)
Hprt-, TK, Hprt TK-
Hprt-, TK, Hprt TK-
Correlate identified chromosome loss With loss of
phenotypic trait (isozyme, DNA sequence, etc.)
2
More mutant selection methods

• 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

3
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)
4
Mike Wigler
Richard Axel
Saul Silverstein
History discovered for practical use at Columbia
(PS Wigler Axel and Silverstein)
5
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 -gt 1000 colonies
could be much less for certain types of cells
6
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 (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 transducing pathway for sensing growth factors
7
Recombination gene targeting Mitotic
recombination between homologous chromosomes
relation to cancer through the loss of tumor
suppressor genes LOH loss of homozygosity WT
/ ? mutation ? /- (WT phenotype) ? (LOH
via homologous recombination in G2 or chromosome
loss and duplication)? -/- (mutant phenotype
revealed) Recombination of transfecting
genes homologous vs. non-homologous
recombination. Gene conversion vs. reciprocal
recombination. Recombination between tandem
inserts (higher freq).
8
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) 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
9
Double knockout of the ASF gene, a vital gene, by
homologous recombination
Chicken DT40 cells

ASF-
neo
Tet-off promoter
pur
neo
ASF-
neo
tet
pur
ASF-
pur
X
Cell dies without ASF
cell viable
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.
10
Gene amplification for high level production in
CHO dhfr- cells.
DHFR system (dihydrofolate reductase)
Selection for resistance to marginal levels of
methotrexate
DHFR
DHFR
Folate
tetrahydrofolate
dihydrofolate
Glycine Purine nucleotides (AMP and
GMP) Thymidylic acid (TMP)
FH4
FH2
Resistance 1) Methotrexate permeation mutants
(incl. MDR, increased efflux)) 2) Altered DHFR
with lower MTX binding affinity 3) Overproduction
of DHFR protein
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Gene amplification Historically Methotrexate
resistance (Littlefield) High dihydrofolate
reductase (DHFR) enzyme activity, protein,
protein synthetic rate, translatable mRNA. mRNA
level, DNA level. (Alt and Schimke) Homogeneousl
y staining, expanded chromosomal regions (HSRs)
(Biedler) HSR dhfr genes (Nunberg, Chasin)
Double minute chromosomes (Gilbert). Amplicons.
Big (300 KB). Can shrink, migrate (Hamlin).
13
Gene amplification
HSR Homogenously staining region
Nunberg et al. PNAS)
(Schimke, Sci. Amer.)
14
Gene amplification
Homogeneously staining region FISH, here
15
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
16
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
17
Fred Alt
Geoff Wahl
George Stark
18
Reduction of folate to tetrahydrofolate
19
Biosynthesis of glycine
20
Biosynthesis of TMP
21
Biosynthesis of purine nucleotides
22
DHFR- cells require G,H,T
23
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)
24
Some other amplifiable genes
25
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

26
Linked amp
CHO cells
27
Co-amp1
28
Co-amp3
(with or without pre-ligation)
29
kaufman
30
Co-amp2
31
Co-amp4
32
Amplification protocol
33
G.E.N. 11/2005
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35
Position effect (Reff- IDEC) on high
production Expression level is influenced by the
position of integration (in transgenic mice and
transfected cells ) Euchromatin vs.
heterochromatin, gradation, chromatin
organization, proximity of enhancers M. Reff
(patent) Screen for a high producer site among
many transfectants. Integrated gene is linked to
1/3 of a neo gene (3 exons total), and several
selectable markers including dhfr (amplifiable)
. Use this transfectant as the host for YFG
linked within (why?)the other 2/3 of the neo
gene (between 2 introns). Overlapping neo
sequences target homologous recombination. Select
for G418 resistance (reconstruction of the neo
gene) Drug-resistant colonies carry YFG at the
hot spot for production,within an intron of the
neo gene. Homologous recombination frequency is
low (10-7), but you only need one good
transfectant. Amplify with MTX / dhfr.
36
CMV-MCS
dhfr
lucD
GUS
HisD
neo3
Marker, integrated first.
neo1
neo2
CD20
Targeting plasmid
37
Reff patent 2002 (IDEC-Biogen) Exploits position
dependencefor high expression level
dhfr
Neo exon 3 with 3ss
Histidinol dehydrogenase gene
Transfectants selected in histidinol resistance
(for HisD gene) AND minus purine thymidine
(for the dhfr gene)
38
Histidinol dehydrogenase detoxifies histidinol,
confers histidinol resistance
protein synthesis
inhibits protein synthesis (charged to tRNA but
cannot be transferred so truncates)
39
Neo neomycin phosphotransferase gene,
detoxifies neomycin in bacteria, G418 in higher
organisms
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41
Low freq event lt 10-6)
From Molly
Select in G418 (neoR) for reconstruction of the
neo gene
42
Ig promoters
splicing
Mab genes are expressed from within the neo
gene Advantage? Tightly integrated, never lost
in a neoR cell. Disadvantage? Complex
transcription, competition?
43
More amplification by 2 sequential amplifications
(R. Kaufman)

• Dhfr MTX
• Adenosine deaminase (ADA) deoxycoformycin
• AAU medium
• adenosine (requires ADA)
• alanosine (inhibits purine synthesis)
• uridine (counteracts high adenosine toxicity
  obscure)
• deoxycoformycin (inhibits ADA
  gradual increase of dose)

After a maximum is reached with MTX, bring in YFG
again linked to ADA and go through another whole
amplification regimen
44
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 gln-free
medium. Inhibit GS with methionine sulfoximine
(gln analog). Select for GS overproducers.
(amplification of the GS cDNA gene and linked
Mab genes) Proprietary (Lonza Biologics).
45
Industry growth

• Global biopharmaceutical sales
• Year Sales Growth
• 2000 23 16
• 2001 27 19
• 2002 33 23
• 2003 40 21
• 2004 48 20

• Growth of biopharmaceuticals vs. pharmaceuticals
• 2000 2001 2002 2003 2004
• Global pharmaceutical sales 354 392 430 492 550
• Growth () 6.6 10.7 9.7 14.4 11.8
• Biotech share 6.4 6.9 7.6 8.1 8.7
• GEN Nov. 2005

46
Some marketed recombinant proteins Erythropoietin
(Epogen, Procrit) JJ, Amgen Tissue
plasminogen activator (TPA) Genentech Growth
Hormone (Genentech) Insulin (Genentech) Beta-in
terferon (Avonex) Biogen-IDEC Alpha-interferon
(IntronA) Schering-Plough Neupogen
(Amgen) Etanercept TNF receptorIgG (Enbrel)
Amgen Monoclonal antibodies (mAbs)
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48
20,000 liter mammalian cell ferment or - Lonza
Biologics - Portsmouth, NH
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50
High level production in mammalian cells Do the
math

• Reff 55 pg/cell/day
• Max cell density 2 x 106/ml ?
• So 2 x 109 cells/L
• Therefore 55 x 10-12 g/cell/day x 2 x 109
  cells/L
• 110 x 10-3 g/L/day
• 0.1 g/L/day
• Lonza (contract manufacturer) claims (2004)
• 2.8 g/L yield
• OK, if accumulate secreted protein for 28 days at
  0.1 g/L/day.
• (long, but not unreasonable)
• 30,000 L reactor
• 30,000 L. X 2. 8 g/L. 84 kg in 28 days,
• x 12 1008 kg/year 1,000,000 g/year
• One MAb dose 500 mg 0.5 g
• 1,000,000/0.5 2 million doses per reactor per
  year.
• 6 doses per patient per year?