Rice paper cont.

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Rice paper cont.
TPA Tissue plasminogen activator, dissolves
clots Problem Cleared quickly from bloodstream
by liver Bind to hepatocytes in liver via TPAs
kringle domain Want to isolate a TPA mutant
protein with less affinity for hepatocytes Must
be still enzymatically active of course.
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Goal to improve tissue plasminogen activator as
a therapeutic clot-busting treatment Means
Reduce or eiminate the binding of tPA to liver
cells, as this clears it from the blood Authors
here use a mammalian cells as the carrier of the
DNA and the cell surface as a display site.
Display was via a fusion protein to a membrane
anchor protein, DAF (peptide, really). DAF
decay accelerating factor What did they
do? Cassette mutagenesis. What region? 333 bp K1
(kringle-1), known to bind the MAb387, which
competes for hepatocyte binding (so assuming it
is the same target epitope). How did they get
kringle mutated? Error-prone PCR How did they
isolate just the kringle 1 region? PCR
fragment. How did they get the mutagenized
fragment back in? Introduced restriction sites at
the ends, w/o affecting the coding.
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What did they put the mutagenized fragment
into? DAF TPA fusion protein geneHow did they
get it into into cells? Electroporation What
cells did they use as hosts? 293 carrying SV40
large T antigen How many copies per cell. And
why is that important? One, by electroporation
at low DNA concentration. In a transient
transfection! Binding is dominant. Lack of
binding (what they are after) is recessive. How
did they select cells making MAb387-non-binding
TPA? FACS Recover cells that bind fluorescent
mAb vs. protease domainbut low binding to
fluorescent mAb vs. kringle domain
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Tracked down vector contains SV40 ori and is
transfected into 293 cells making SV40 T-antigen.
So plasmid replicates during the transient
transfection ? higher signal.
5
,
Sort the cells with low fluorescence
For reiteration of the process
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How did they recover the plasmid carrying the
mutant TPA gene from the selected cells? Hirt
extraction Like a plasmid prep, lyse cells
gently, high MW DNA entangles and forms a
clot. Centrifuge. Chromosomal DNA ? soft
pellet plasmid DNA circles stay in supernatant.
Then re-transfect, re-sort in FACS. After 2
sorting rounds, test individual E. coli clones
60 are binding-negative.
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MAb to protease domain
enriched
Collect these
No good
good
good
good
good
Low kringle-1 reactivity
MAb to kringle-1 domain
FITC fluorescein reagent. PE
phycoerythrin (fluorescent protein)
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Hepatoma cell binding. How? Clone mutated
regions into regular TPA gene for testing (no
DAF, protein now secreted) Label WT TPA with
fluorescein (FITC, conjugated chemically) Mix
with hepatoma cells and analyze on a flow
cytometer (FACS w/o the sorter part). See
specific and non-specific binding. Subtract
non-specific binding the amount not competed by
excess un-labeled wt TPA.
FITC fluorescein isothiocyanate
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Hepatoma cell binding assay measure competition
for binding of fluorescently labeled WT TPA
Binding assay, initial condition
No competitor
WT
Compete. So still bind.
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Mammalian cell
genetics Introduction Genetics as a subject
(genetic processes that go on in somatic
cells that replicate, transmit, recombine,
and express genes) Genetics as a tool. Most
useful the less you know about a process. 4
manipulations of genetics 1- Mutation in
vivo (chance selection, usually) targeted gene
knock-out or
alteration in vitro site directed or random
cassette 2- Mapping Organismic mating
?segregation, recombination (e.g., transgenic
mice) Cell culture
cell fusion segregation radiation hybrids
FISH 3- Gene juxtaposition (complementation)
Organisms matings ? phenotypes of
heterozygotes Cell culture cell fusion ?
heterokaryons or hybrid cells 4- Gene transfer
transfection
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Mammalian cell genetics Advantages of cultured
cells (vs. whole organism) numbers,
homogeneity Disadvantages of cultured mammalian
cells limited phenotypes limited
differentiation in culture (but some phenotypes
available) 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 Variants
could be due to Stable heritable alterations in
phenotype that are not due to mutations
heritable switches in gene regulation (we dont
yet understand this). DNA CpG methylation,
histone acetylation / de-acetylation Diploidy.
Heteroploidy. Haploidy. The problem of diploidy
and heteroploidy Recessive mutations (most
knock outs) are masked. (cf. e.g., yeast, or C.
elegans, Dros., mice) F2 ? homozygotes)
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Solutions to diploidy problem Double mutants
(incl. also mutation segregation, or mutation
homozygosis(rare but does occur) Heavy
mutagenesis, mutants/survivor increases but
mutants/ml decreases. How hard is it to get
mutants? What are the spontaneous and induced
mutation rates? (loss of function
mutants) Spont 10-7/cell-generation
Induced 2 x 10-4 to 10-3 /cell (EMS,
UV) So double knockout could be 0.00072 5X10-7.
One 10cm tissue culture dish holds 5x106
cells. Note Same considerations for creation
of recessive tumor suppressor genes in cancer
requires a double knockout. But there are lots of
cells in a human tissue or in a mouse. RNAi
screen, should knock down both alleles Transfect
with a library of cDNA fragments designed to
cover all mRNAs. Select for knockout phenotype
(may require cleverness). Clone cells and recover
RNAi to identify target gene. A human near
haploid cell strain. Use of it Science, 326
1231-1235 (2009) EMS ethyl methanesulfonate
ethylates guanine UV (260nm) induces dimers
between two adjacent pyrimidines on the same DNA
strand
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L
R
R
L
Homozygosis Loss of heterozygosity (LOH)by
mitotic recombination between homologous
chromosomes (rare)
M i t o s i s
-
-
-


2 heterozygotes again
L
R
L
R
L
R
R
L
or
-
-


-
-


Paternal Chr. 4, say
-
Maternal Chr. 4
-
Recombinant chromatids


After homologous recombination (not sister
chromatid exchange)
Heterozygote
1 homozygote /1 homozygote -/-
Recessive phenotype is unmasked
a mechanism of homozygosis of recessive tumor
suppressor mutations in cancer
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Mutagenesis (induced general mutations, not site
directed) 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 Dominant vs. recessive
mutations Dom. are rare (subtle change in
protein), but expression easily observed,
Recessives are easier to get (whatever KOs the
protein function), but their expression is masked
by the WT allele.
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• Categories of cell mutant selections
• Example
• Auxotrophs purine-
• Drug resistance Dominant ouabainR,
  alpha-amanitinR Recessive 6TGr, BrdUr
• Antibodies vs. surface components MHC-
• Visual inspection G6PD-, Ig IP-
• FACS fluorescence-activated cell sorter DHFR-
• Brute force IgG-, electrophoretic shifts
• Temperature-sensitive mutants 3H-leu resistant

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(drugs, in italics)
PRPP phosphoribosyl pyrophosphate
FH4tetrahydrofolate
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Test yourself Fill in the boxes Grow () or not
grow(-) Click here for the answers
Growth pattern examples
GHT glycine, hypoxanthine, and thymidine A
adenine H hypoxanthine G glycine TG
6-thioguanine (G analog) DAP diaminopurine
(A analog) MTX methotrexate (DHFR
inhibitor) DHFR dihydrofolate reductase HPRT
hypoxanthine-guanine phosphoribosyltransferase APR
T adenine phosphoribosyltransferase
Only mutation GHT -GHT -GHT 6TG -GHT DAP -H GT MTX -H GT MTX A -H GT MTX Guanine -H GT MTX H
WT
APRT-
HPRT-
DHFR-
in italics
-


-
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Cell mutant types

• 1. Auxotrophs (BrdU reverse selection, not
  discussed)
• 2. Drug resistance (dominants or recessives)
• 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 (Ig anti-Ig IP)
• FACS fluorescence-activated cell sorter (cell
  surface antigen or internal ligand binding
  protein)
• Brute force (clonal biochemical analysis, e.g.,
  electrophoretic variants (e.g., Ig, isozymes))
• MHC major histocompatability locus or proteins
• G6PD glucose-6-phosphate dehydrogenase

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Cell fusion (for gene juxtaposition, mapping,
protein trafficking, etc. ) Fusogenic agents
PEG, Sendai virus (syncytia promoting, as
HIV). Heterokaryons (2 nuclei), no cell
reproduction (limited duration). (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) allows
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, (available 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
Heterokaryon (or, alternatively, homokaryon)
HAT medium
Hprt-, TK Hprt TK-
Hybrid cell
HAT
Cell cycle, Nuclear fusion, Mitosis, Survival, rep
roducton
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 cells examples of use gene mapping
(synteny) gene regulation (extinction) Complementa
tion (pyrimidine path)
Synteny genes physically linked on the same
chromosome are syntenic.
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Frye and Edidin, 1970 Use of cell fusion and
heterokaryons to measure the difusio of membrane
proteins
Complete mixing in lt 40 min. No diffusion at low
temperature (lt15-20 deg)
http//www.erin.utoronto.ca/w3bio315/lecture2.htm
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Complementation analysis
Mutant parent 1
Mutant parent 2
Mutant parent 1
Mutant parent 2
gly2-


gly3-
gly1-
gly1-
Cell fusion
Cell fusion
Hybrid cell
Hybrid cell
glyA- glyA-
glyA- glyB-
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 (hybrids)
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 fragments the human donor cell
chromosomes After fusion, some fragments are
integrated into the rodent chromosomes. 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 integrated together the
closer the linkage.,
x
Select for a human gene (e.g., hprt) to
eliminate rodent parental cells (e.g., x hprt-)
Irradiated human cells die
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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)