W30344034 Mammalian cell genetics lecture

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W3034/4034 Mammalian cell genetics
lecture Intro Genetics as a subject
(genetic things that go on in somatic cells
replicate, transmit, recombine, express
genes) Genetics as a tool. Best when you know
nothing. 4 manipulations of genetics 1-
Mutation in vivo (selection, usually) gene
knock-out, in vitro (site directed or random
cassette) 2- Mapping, organismic matings,
recombination cell
fusion/segregation, radiation hybrids,
FISH 3- Gene juxtaposition (complementation)
matings -gt heterozygotes cell fusion -gt
heterokaryons or hybrid cells 4- Gene transfer
transfection
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Advantages of cultured cells numbers,
homogeneity Disadvantages of cultured mammalian
cells Limited phenotypes, limited
differentiation in culture (but phenotypes OK),
no sex (cf. yeast)
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 immortalized, plus
"transformed, plus have abnormal karyotypes)
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Mutation in cultured mammalian cells Problem of
epigenetic change. Variants vs. mutants, Stable
heritable alterations in phenotype that are not
due to mutations heritable switches in
gene regulation (?) DNA CpG methylation. Diploi
dy. Heteroploidy. Haploidy. The problem of
diploidy and heteroploidy (cf. yeast, or even C.
elegans, Dros.) Recessive mutations (most knock
outs) are masked.
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Solutions Dominant mutations only (too
limited) Haploid cells (hasn't worked,
although frogs, and modest chromosome reductions
in CHO) Haploid genes (XY) or functionally
haploid (XX, allelic exclusion)) Heterozygous
loci (rare, despite CHO reputation) Double
mutants (incl. mutation segregation, or
mutation homozygosis rare but can be
done) 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.
Spont 10-7/cell-generation Induced
as high as 10-3 /cell (EMS, UV)
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Mutagenesis. Chemical and physical agents
MNNG point mutations (single base
substitutions) EMS
Bleomycin small
deletions UV mostly point mutations but also
large deletions Ionizing radiation (X-,
gamma-rays) large deletions, rearrangements Dosa
ge kill 90 Expression period dilute out
WT molecules (pre-existing protein. mRNA)
Metabolic cooperation WT toxic product can be
transferred.. Dominant vs. recessive mutations
Dom rare (subtle) but expressed, Recessives
easy but masked. Mutagen specificity ( a
particular base or base combo). Mutational
spectra (hot and cold spots). 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)
• Amino acid biosynthesis auxotrophs
• Auxotrophs BUdR (BrdU) Kao and Puck. Kill
  growing cells. Penicillin analogy.
• Amino acid, nucleotide biosynthetic
  pathways.
• 2. Drug resistance see sheet
• 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)
• C. Permeation changes influx blocked or efflux
  increased. (MDR, P-glycoprotein)
• D. Improved de-toxification via chelation,
  covalent modification,
• or overproduction of target (dhfr
  MTX-resistance)
• E. Receptor deficiency
• Glucocorticoid (steroid) receptor-resistant to
  GC killing of lymphoid lines.

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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, protein
trafficking, mapping) 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). Complementation (e.g., auxotrophs
with same requirement) Dominance vs.
recessiveness. Mapping chromosome assignment.
Synteny. Radiation hybrids linkage analysis
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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 discovered for practical use at
Columbia (PS Wigler Axel and Silverstein)
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Transient transfection vs. permanent cloned
genes 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
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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 (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
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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, or loss and
duplication ? -/- Recombination of transfected
genes homologous vs. non-homologous
recombination. Gene conversion vs. reciprocal
recombination. Recombination between tandem
inserts (higher freq) Gene knockouts via
homologous recombination. ES cells and
transgenic mice. Selection for homologous
recombinants via the loss of viral TK genes
(Capecchi). Allele replacements in cultured cell
lines (e.g., APRT). Little work in cultured
lines myc double K.O. viable, sick
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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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Loss of heterozygosity by mitotic recombination
between homologous chromosomes (rare)
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1 homozygote 1 homozygote -
or
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Heterozygote
After homologous recombination
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2 heterozygotes again
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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. In nature rDNA in oocytes,
Drosophila chorion genes. In medicine
chemotherapy resistance (MDR, P-glycoprotein)
cancer (myc, ras) In biotechnology high
level recombinant protein production in mammalian
cells