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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 TPA’s kringle domain
Want to isolate a TPA mutant protein with less affinity for hepatocytes
Must be still enzymatically active of course.
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.
What did they put the mutagenized fragment into?
DAF – TPA fusion protein gene
How 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 domain
but low binding to fluorescent mAb vs. kringle domain
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.
,
Sort the cells with
low fluorescence
For
reiteration
of the
process
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.
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)
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
Hepatoma cell binding assay:
measure competition for
binding of fluorescently
labeled WT TPA
Binding assay,
initial condition
Can’t compete (good)
No competitor
WT
Compete.
So still bind.
But still have
protease activity
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
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)
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 don’t 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)
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
L
R
Homozygosis:
Loss of heterozygosity (LOH)
by mitotic recombination between
homologous chromosomes (rare)
L
R
L
M
i
t
o
s
i
s
R
--
R
L
+
-
+
2 heterozygotes again
L
R
L
R
or
+
+
-
-
Paternal
Maternal
Chr. 4, say Chr. 4
Heterozygote
+
-
+
-
Recombinant
chromatids
After homologous recombination
(not sister chromatid exchange)
Recessive phenotype is unmasked
+
+
-
1 homozygote +/+
1 homozygote -/-
= a mechanism of homozygosis of recessive tumor suppressor mutations in cancer
-
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 KO’s the protein function), but their
expression is masked by the WT allele.
Categories of cell mutant selections
Example
purine-
•
Auxotrophs
•
Drug resistance
Dominant
Recessive
ouabainR, alpha-amanitinR
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
Purine biosynthesis, salvage pathways, and inhibitors
Adenine(A)
(diaminopurine)
(8-azaadenine)
Methotrexate
(=amethopterin)
(~aminopterin)
Folate
Adenosine
APRT
Adenosine
kinase
FH4
AMP
Nuc. Acid
GMP
Nuc. Acid
Glycine
Thymidine (T)
Alanosine Adenylosucc.
PRPP +
glutamine
Azaserine
Glutamine
IMP
HGPRT
XMP
Hypoxanthine
(H)
XGPRT
(Eco gpt)
Xanthine
(X)
Mycophenolic
acid
Code:
Biosynthesis;
Salvage enzymes
Inhibitors
(drugs, in italics)
Analogs (iytal.)
PRPP = phosphoribosyl pyrophosphate; FH4=tetrahydrofolate
HGPRT
Guanine
(6-thioguanine)
(8-azaguanine)
Test yourself: Fill in the boxes
Grow (+) or not grow(-)
Click here for the answers
Growth pattern examples
Purine biosynthesis, salvage pathways, and inhibitors
Adenine(A)
(diaminopurine, DAP)
(8-azaadenine, 8AA)
Methotrexate
(=amethopterin) Folate
(~aminopterin)
Adenosine
APRT
Adenosine
FH4
kinase
Nuc. Acid
AMP
Glycine
Thymidine (T)
Adenylosucc.
Alanosine
GHT = glycine, hypoxanthine, and thymidine
PRPP +
glutamine
A = adenine
H = hypoxanthine
G = glycine
Azaserine
TG = 6-thioguanine (G analog)
Glutamine
DAP = diaminopurine (A analog)
MTX = methotrexate (DHFR inhibitor)
DHFR = dihydrofolate reductase
HPRT = hypoxanthine-guanine phosphoribosyltransferase
APRT = adenine phosphoribosyltransferase
Only
mutation
WT
APRTHPRTDHFR-
+GHT
+
-GHT
-GHT
+6TG
IMP
HGPRT
XMP
Hypoxanthine XGPRT
(H)
(Eco gpt)
-GHT +
DAP
Mycophenolic Xanthine
(X)
-H +GT
+MTX
-
GMP
Nuc. Acid
HGPRT
Guanine
(6-thioguanine, 6TG)
(8-azaguanine, 8AG)
-H +GT
-H +GT
in italics
+MTX
+MTX +
Guanine
+A
+
-
-H +GT
+MTX
+H
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)
6. FACS = fluorescence-activated cell sorter (cell surface antigen or internal
ligand binding protein)
7. 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. IP = immunoprecipitate
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)
Cell fusion
Hprt+, TK-
+
Parental cells
Hprt-, TK+
HAT-
HAT-
PEG (polyethylene glycol, mw ~ 6000
Sendai virus, inactivated
Cell fusion
Hprt-, TK+
Heterokaryon
(or, alternatively, homokaryon)
Hybrid cell
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)
Synteny = genes physically linked on
the same chromosome are syntenic.
HAT
medium
HAT
+
Cell cycle,
Nuclear fusion,
Mitosis,
Survival,
reproducton
Hprt-, TK+,
Hprt+ TK-
Hybrid cells: examples of use:
gene mapping (synteny)
gene regulation (extinction)
Complementation (pyrimidine path)
Frye and Edidin, 1970:
Use of cell fusion and heterokaryons to measure the difusio of membrane proteins
Complete mixing in < 40 min.
No diffusion at low temperature (<15-20 deg)
http://www.erin.utoronto.ca/~w3bio315/lecture2.htm
Complementation analysis
Mutant parent 1
Mutant parent 2
gly2-
+
gly1-
Mutant parent 1
Cell fusion
+
Glycine-free medium: No growth
No complementation
same gene
(named glyA)
gly3-
gly1Cell fusion
Hybrid cell
glyA- glyA-
Mutant parent 2
Hybrid cell
glyA- glyB-
Glycine-free medium: Yes, growth
Yes, complementation
different genes genes
(named glyA and glyB)
Mapping genes to chromosomes (hybrids)
Hprt- x tk- Hybrid cell (Human x Rodent)
Hprt-, TK+,
Hprt+ TK-
Reduced hybrid
Spontaneous
chromosome
loss (human ~
preferentially lost)
Just passage and wait
Hprt-, TK+,
Hprt+ TK-
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).
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
Irradiated human cells die
Select for a human gene
(e.g., hprt) to eliminate rodent
parental cells (e.g., x= hprt-)
Ted Puck: mutagenesis;
auxotrophic mutants in CHO
cells (U. Colo.)
nuclear-cytoplasmic shuttling
in heterokaryons (Penn)
Helen Blau:
turning on muscle
genes in
heterokaryons
(Stanford)
Frank Ruddle:
mapping by
chromosome
segregation from
cell hybrids.(Yale)
Mary Weiss:
turning off
differentiation genes
in cell hybrids
(Institut Pasteur)
Michael Edidin:
2-D diffusion of
proteins in the cell
membrane
in heterokaryons
(Johns Hopkins)