Induction and Isolation of mutant mammalian cells

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• Induction and Isolation of mutant mammalian cells
• Diploid for most genes means loss of function
  mutations are masked (recessive).
• Twp events are needed to unmask (express)
  recessive alleles.
• Double mutation A/A ? a/a. Feasible with good
  mutagens. E.g.,
• at a freq. of 2x10-4 each ? 2 x 10-4 at first
  allele x 2 x 10-4 at second allele x 2 ways
  (orders) to do this ?8 x 10-8
• If 10 survival of mutagen treatment, need to
  mutagenize 2 x 108 cells to get one mutant
• 2 x 107 survivors ? 1.6 mutants. Just feasible.
• But the two events need not be point mutations.
• One event could be more a radical change at a
  higher frequency chromosome loss, mitotic
  recombination
• Same considerations apply to the unmaskinig of
  recessive loss of 2 copies of wild type tumor
  suppressor genes ini cancer.

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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 affecting 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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Isolation of auxotrophic mammalian cell mutants

• Mutagenize a culture of cells grown in a rich
  medium.
• Then let grow for a while to allow expression of
  mutant phenotypes.
• Starve cells for a nonessential nutrient, e.g.,
  glycine,
• Wild type cells continue to grow
• Glycine auxotrophs stop growing, stop DNA
  synthesis, start to die, perhaps.
• After 24 hours, add BrdU, a thymidine analog (Br
  in place of the methyl group)
• Growing cells incorporate BrU into their DNA
  glycine auxotrophic mutant cells do not.
• After 24 more hours, irradiate cells with 313 nm
  light. Normal DNA does not absorb light of this
  wavelength, BrdU-containing DNA does absorb, so
  DNA is damaged and WT cells die.
• Change to a rich medium (containing glycine,
  here) and let the mutant cells grow into
  colonies.
• Test individual clones of cells for their ability
  to grow or glycine.

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Some auxotrophs of Chinese hamster cell
lines Glycine Pyrimidine (uridine)
Thymidine Purine (hypoxanthine) Inositol Chol
esterol Choline
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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. Altered enzyme protein. Enzyme target
  becomes a better discriminator
• (ouabain NaK ATPase pump
  alpha-amanitinRNA Polymerase II)
• C. Permeation changes influx blocked or efflux
  increased. (MDR via P-glycoprotein)
• D. Improved de-toxification via chelation of
  drug, covalent modification of drug,
• or overproduction of its target (dhfr
  MTX-resistance via overproduction neoR
  neomycin phosphotransferase)

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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. Altered enzyme protein. Enzyme target
  becomes a better discriminator
• (ouabain NaK ATPase pump
  alpha-amanitinRNA Polymerase II)
• C. Permeation changes influx blocked or efflux
  increased. (MDR via P-glycoprotein)
• D. Improved de-toxification via chelation of
  drug, covalent modification of drug,
• or overproduction of its 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. (cell
  surface Ag less commonly internal fluorescent
  dye binding)
• 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 LDH
  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 proportion compared to
heterokaryons. Complementation (e.g., auxotrophs
with same requirement) Dominance vs.
recessiveness. Chromosome 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
HAT medium
Hprt-, TK, Hprt TK-
HAT
Heterokaryon (or, alternatively, homokaryon)
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
Mutant parent 1
Mutant parent 1
Parental cells
Mutant parent 2
Mutant parent 2


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-
Hprt-, TK, Hprt TK-
Just passage and wait
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).
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Radiation hybrids
Ionizing radiation frgaments the human donor cell
chromosomes After fusion, some fragments are
integrated intot he rodent cromosomes. Checking
these reduced hybrids for human markers ( DNA
restriction fragments, PCR products, or isozymes)
allows conclusion about genetic linkage, the more
often two markers are intergrated together the
closer the linkage.,
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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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Gene transfer in cultured mammalian
cells Transfection agents DEAE-dextran (toxic,
OK for transient) CaPO4 (co-precipitate) Electropo
ration (naked DNA, high quick voltage ? transient
holes) Lipofection (multilamellar
liposomes) Polybrene (cationic polymer,
hydrocarbon stretches with N charges, Br-
counterion) 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 (very 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)
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Transfections 2 types
Transient 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 number). i.e., 106
cells ? 1000 colonies could be much less for
certain types of cells.
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Got this far
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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
Transformed Mouse 3T3 cells transfected with an
EGFreceptor gene
Mouse 3T3 cells
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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)
See figure at right. Non-homologous
recombination favors ends tk is inserted,
conferring sensitivity to the drug gancyclovir
(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 sequential 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 APRT adenine
phosphoribosyltransferase ASF alternative
splcing factor
Resistant to gancyclovir
Die in gancyclovir
HSV-TK gene is removed during homologous
recombination, left joined during non-homologous
recombination. Unlike mammalian TK, HSVTk
converts gancyclovir to a toxic product HSV
Herpes simplex virus tk thymidine kinase FIAU
equivalent to gancyclovir, today
M. Capecchi, Nature Medicine  7, 1086 - 1090
(2001) Generating mice with targeted mutations
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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.
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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 can occur via 3 different
mechanaisms 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.
Large (300 KB). Can shrink, migrate (Hamlin).
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Gene amplification
HSR Homogenously staining region
Nunberg et al. PNAS 1980
(Schimke, Sci. Amer.)
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Gene amplification
Homogeneously staining region FISH, here
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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
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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). Mutation of the p53 gene allows it. 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
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Fred Alt
Geoff Wahl
George Stark
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Reduction of folate to tetrahydrofolate
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Biosynthesis of glycine
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Biosynthesis of TMP
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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. These
cells 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
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Co-amp1
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Co-amp3
(with or without pre-ligation)
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kaufman
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Co-amp2
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Co-amp4
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Amplification protocol