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Categories of cell mutant selections

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GHT DAP GHT. Only mutation. GHT = glycine, hypoxanthine, and thymidine. A = adenine ... H DAP H. Only mutation. Originally intended table: Red = corrected ... – PowerPoint PPT presentation

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Title: Categories of cell mutant selections


1
  • Categories of cell mutant selections
  • Exploitable metabolic pathways
  • Purine and pyrimidine biosynthesis auxotrophs
  • (auxotrophs require a nutrient in the medium that
    the WT doesnt)
  • 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 have been isolated (most
    in CHO)
  • 2. Drug resistance see sheet on nucleotide
    metabolism
  • A. Mutant lacks toxifying enzyme (a major
    class)
  • e.g., HPRT (TGR), APRT (DAPR, 8-azaAR), TK
    (BrdUR)
  • B. Enzyme target becomes a better discriminator
  • (ouabain NaK ATPase pump a-amainitin
    RNA Pol II)
  • 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)

2
in italics
3
Originally intended table
Growth pattern examples
GHT glycine, hypoxanthine, and thymidine A
adenine G guanine
in italics

-
-
-


-





-
-
-
-
-

-
-
-
-
-
-
Red corrected from original graphic
4
HAT hypoxanthine amethopterin thymidine
-


-
-
-


-

-

5
Cell mutant types cont.
  • 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)
  • MHC major histocompatability locus or proteins
    G6PD glucose-6-phosphate dehydrogenase LCH
    lactate dehydrogenase Ig immunoglobulin

6
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., studied
membrane fluidity, nuclear shuttling, gene
activation (myoblasts) Hybrids (nuclei fuse,
some cells (minority) survive and reproduce).
Small of heterokaryons. Complementation (e.g.,
auxotrophs with same requirement) allos
selection Dominance vs. recessiveness can be
tested. Chromosome loss from hybrids ? Mapping
chromosome assignment. Synteny. Radiation
hybrids linkage analysis (sub-chromosomal
regional assignments).
PEG polyethylene glycol, (1000 to 6000 MW)
7
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-
Heterokaryon use examples membrane dynamics
(lateral diffusion of membrane proteins) shuttling
proteins (e.g., hnRNP A1 ), gene regulation
(e.g., turn on myogenesis)
Hybrid cell
Hybrid cell use examples gene mapping
(synteny) gene regulation (extinction) Complementa
tion (pyrimidine path)
Synteny genes physically linked on the same
chromosome are syntenic.
8
Complementation analysis
Parental cells
Mutant parent 1
Mutant parent 1
Parental cells
Mutant parent 2
Mutant parent 2

glyA2-

glyB1-
glyA1-
glyA1-
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)
9
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).
10
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.,
Select for a human gene (E.g., hprt) to eliminate
rodent parental cells
Irradiated cells die
11
Ted Puck mutagenesis auxotrophic mutants in CHO
cells (U. Colo.)
Mary Weiss turning 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 membrane in heterokaryons (Johns Hopkins)
Frank Ruddle mapping by chromosome segregation
from cell hybrids.(Yale)
nuclear-cytoplasmic shuttling in heterokaryons
(Penn)
12
DNA transfection
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). Co-integration of high MW DNA .
Can 2000 KB. Separate plasmids transfected
together ? same site (co-integration). Separate
transfections ? 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)
DEAE diethyl-amino-ethyl (positively charged)
13
Mike Wigler
Richard Axel
Saul Silverstein
History discovered for practical use at Columbia
(PS Wigler Axel and Silverstein)
14
Transient transfection vs.
permanent cloned genes Unintegrated DNA
chromosomally
integrated. Unnatural?
position effects
? Super-physiological expression (so
analyze a pool of many to average) levels
(per transfected cell) ? Transient -gt 10-50
transfection efficiency (stain) Permanents more
like 0.001 transfectants per µg DNA per cell
(high). i.e., 106 -gt 1000 colonies could be
much less for certain types of cells
15
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
16
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 (rare) vs. non-homologous
(common) recombination.
17
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
18
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.
19
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
20
Gene amplification dhfr Historically
Methotrexate resistance MTX inhibits
dihydrofolate reductase (DHFR) MTX-resistant
cells have (in order of discovery, 1970s) High
DHFR enzyme activity High DHFR protein High
protein synthetic rate High translatable
mRNA High mRNA level (by hybridization) High DNA
level. Homogeneously staining, expanded
chromosomal regions (HSRs HSRs are the locatin
of the high number of dhfr genes. Double minute
chromosomes are an occasional alternative
form. Amplicons (distance between repeated
genes) are large (300 KB). (dhfr gene 25 kb)
HSRs can shrink, migrate.
21
Reduction of folate to tetrahydrofolate
MTX
22
Methotrexate
23
Gene amplification
HSR Homogenously staining region
Nunberg et al. PNAS 1980
(Schimke, Sci. Amer.)
24
Gene amplification
Homogeneously staining region FISH, here
FISH fluorescent in situ hybridization
25
Original locus?
HSR ? dmin upon DS break induced by a homing
endonuclease (I-SceI).
Restriction-type enzyme with a very long
recognition sequence ( 20 bp)
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 meganuclease I-SceI
expression or fragile site activation leads to
formation of double minutes and other chromosomal
rearrangements
26
Some other amplifiable genes
27
Ampification models over-replication, unequal
sister chromatid exchange, breakage and fusion
(Tanaka et al.). Map dhfr amplicons (Schimke,
Hamlin) 300 kb , but wide range Gene
amplification is rare in normal cells (Wahl,
Tslty). p53- mutation allows it. In nature
rDNA in oocytes, Drosophila chorion genes. In
medicine chemotherapy resistance (MDR,
P-glycoprotein, efflux pump) cancer (myc,
ras) In biotechnology high level recombinant
protein production in mammalian cells
28
Some notable gene amplification players
Fred Alt
Geoff Wahl
George Stark
29
Gene amplification for high level recombinant
protein production in mammalian cells.
Principal system dhfr- CHO cells Facilitated
by the availability of DHFR-deficient mutant CHO
cells
CHO dhfr- cells vector with dhfr minigene
YFG
-GHT medium Most cells die. Transfectants live.
gradually increasing concentrations of MTX
Cells with gradually amplified dhfr transgenes
survive. YFG is co-amplified along with the dhfr
minigene.
30
DHFR- cells require G,H,T
and are resistant to tritiated deoxyuridine
X
DHFR- cells selected by their resistance to
radioactive 3H-deoxyuridine 3HdU ? 3HdUMP ?
3H-TMP ? 3H DNA ? death from radioactive
decay. DHFR- cells require glycine, hypoxanthine
and thymidine (GHT). In GHT-free medium CHO
dhfr- cells die, but transfectants that have
received a dhfr minigene, YFG, survive.
31
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 does no seem to
beoperating inthis system.of the GS cDNA gene and
linked Mab genes) Proprietary (Lonza Biologics)
32
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

33
Linked amp
CHO cells
34
Co-amp1
35
Co-amp3
(with or without pre-ligation)
36
kaufman
Y.F.G.
DHFR
Dicistronic mRNA
(ribosome read-through)
Also, later, better dhfr translation using an
IRES Internal ribosome intiation site, Mostly
viral but also some cellular genes In theory, not
an advantage.
Y.F.G.
DHFR
IRES
37
Co-amp2
38
Co-amp4
39
Amplification protocol
Note Process is lengthy and tedious.
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