Mammalian cell genetics continued:

1
Mammalian cell genetics continued Gene
transfer Mammalian expression vectors Homologous
recombination Gene amplification
2
Gene transfer Transient transfection vs.
permanent cloned genes Transient -gt 10-50
transient (stain) Permanents more like 0.001 per
µg DNA per cell (high). i.e., 106 -gt 1000
colonies, could be much lessfor some cells )
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
4

• History
• Infectious virus from viral DNA
• Pagano polio, Graham and van der Eb HSV)
• trans-fection vs. infection (vs.
  transformation)
• Selection for a viral gene
• HSV TK gene from inactivated HSV virions, in TK-
  mouse L-cells (Ltk-) (R. Davidson)
• HSV TK using naked DNA, restriction fragments
  (Wigler).
• Optimize conditions using HSV TK restriction
  fragment (cloning in infancy, 1978)
• Selection for cellular genes
• Apply to total DNA, effect the transfer of a
  cellular TK gene using a total genome (chicken,
  human ? Ltk- cells) (8 KB)
• Next aprt into hamster aprt- cells (2 kb)
• Hprt into hprt- L-cells (40 kb)
• Discovery of a new gene

5
Oncogene discovery 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
(e.g., bladder carcinoma) transfected into
growth-controlled mouse 3T3 cells. Look for
foci (focus). Make a phage lambda library from
growth-transformed transfectant. Screen for human
Alu repeat by colony hybridization. Verify that
a cloned DNA yields high frequency of
focus-forming transfectants. Identify cDNA
froagment by hybridization to Southern
blots. Sequence. Identify gene a dominant
oncogene. Ras, a signaling protein (in
transducing pathway for sensing growth factors
6

• Gene transfer
• from total DNA
• from cloned DNA (pure)
• from cloned cDNA libraries
• Mammalian expression vectors
• Need
• Promoter enhancer
• Viral SV40 early or late, RSV-LTR, CMV(early),
  HSV-TK(weak)
• Cellular strong Actin, eIF2 (ICOS)
• Inducible metallothienin, MMTV, Tet(CMV-VP16),
  ecdysone
• 2) PolyA site SV40 late, beta-globin, growth
  hormone
• 3) Intron SV40 early (weak), growth hormone
• often neglected (pure cDNA)
• 4) Selectable marker, for permanents (neo,
  Eco-gpt, pur, hygro, his HolDH)
• Not always necessary on the same plasmid if you
  are satisfied with co-transfection (and usual
  co-intergation).

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 K.O. viable, sick
(J. Sedivy) Splicing factor (ASF) double K.O. in
chick DT40 lymphoid cells (high rate of
homologous recombination (J. Manley) lethal,
but cover with inducible human gene
9
Gene amplification Historically Methotrexate
resistance (Littlefield) High dihydrofolate
reductase (DHFR) enzyme activity, High protein,
high protein synthetic rate, high translatable
mRNA (?1976). (Schimke) mRNA level, DNA level
(1978). Homogeneously staining, expanded
chromosomal regions (HSRs) Biedler Nunberg HSR
dhfr genes. Double minute chromosomes. Amplico
ns. Big (300 KB). Can shrink, migrate.
10
Double minutes
Fragilesite
HSR?
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
11
Models Over-replication (onion skin) Unequal
sister chromatid exchange (after mitosis 2,0
split) Breakage and fusion Breakage near
inverted repeats strand invasion.
Occurences In nature rDNA in oocytes,
Drosophila chorion genes. In medicine
chemotherapy resistance (MDR, P-glycoprotein)
cancer (myc, ras, others) In
biotechnology high level recombinant protein
production in mammalian cells
12
Gene amplification for high level production in
CHO dhfr- cells.
13
Reduction of folate to tetrahydrofolate
14
Biosynthesis of glycine
15
Biosynthesis of TMP
16
Biosynthesis of purine nucleotides
17
DHFR- cells require G,H,T
18
Transfection strategies

• YFG 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

19
Linked amp
20
Co-amp1
21
Co-amp3
22
kaufman
23
Co-amp2
24
Co-amp4
25
Amplification protocol
26
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)
27
Position effect (Reff- IDEC) Expression level is
influenced by the position of integration ( in
transgenic mice and transfected cells
) Euchromatin vs. heterochromatin, gradation,
proximity of enhancers Reff Screen for a high
producer site among many transfectants. Integrated
gene is linked to 1/3 of a neo gene (3 exons),
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.Overlapping neo sequences target
homologous recombination. Select for G418
resistance (reconstrcution 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.
28
CMV-MCS
dhfr
lucD
GUS
HisD
neo3
Marker, integrated first.
neo1
neo2
CD20
Targeting plasmid