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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 t. PA 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 K 1 (kringle-1), known to bind the MAb 387, 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 cells? Electroporation What cells did they use as hosts? 293 carrying SV 40 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 MAb 387 -non-binding TPA? FACS: Recover cells that bind fluorescent m. Ab vs. protease domain but low binding to fluorescent m. Ab vs. kringle domain
Tracked down vector: contains SV 40 ori and is transfected into 293 cells making SV 40 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 Low kringle-1 reactivity good 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 Cp. G 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): F 2 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~ 5 X 10 -7. One 10 cm tissue culture dish holds ~ 5 x 106 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 c. DNA fragments designed to cover all m. RNAs. 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 (260 nm): 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 -- - - Paternal Maternal Chr. 4, say Chr. 4 Heterozygote + - + R L or + + 2 heterozygotes again L + R L R - 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 ouabain. R, alpha-amanitin. R 6 TGr, Brd. Ur • Antibodies vs. surface components MHC- • Visual inspection G 6 PD-, Ig IP- • FACS = fluorescence‑activated cell sorter DHFR- • Brute force Ig. G-, electrophoretic shifts • Temperature‑sensitive mutants 3 H-leu resistant
Purine biosynthesis, salvage pathways, and inhibitors Adenine(A) (diaminopurine) (8 -azaadenine) Methotrexate (=amethopterin) (~aminopterin) Folate Adenosine APRT Adenosine kinase FH 4 AMP Nuc. Acid Glycine Thymidine (T) Alanosine Adenylosucc. PRPP + glutamine Azaserine Glutamine IMP HGPRT Hypoxanthine (H) XMP XGPRT (Eco gpt) Xanthine (X) Mycophenolic acid Code: Biosynthesis; Salvage enzymes Inhibitors (drugs, in italics) Analogs (iytal. ) PRPP = phosphoribosyl pyrophosphate; FH 4=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, 8 AA) Methotrexate (=amethopterin) Folate (~aminopterin) Adenosine APRT Adenosine FH 4 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 +6 TG IMP HGPRT XMP Hypoxanthine XGPRT (H) (Eco gpt) -GHT + DAP Mycophenolic Xanthine (X) -H +GT +MTX - GMP Nuc. Acid HGPRT Guanine (6 -thioguanine, 6 TG) (8 -azaguanine, 8 AG) -H +GT in italics +MTX + Guanine +A + - -H +GT +MTX +H
Cell mutant types: 1. Auxotrophs (Brd. U reverse selection, not discussed) 2. Drug resistance (dominants or recessives) 3. Temperature‑sensitive mutants: cell cycle mutants. Tritiated amino acid suicide (aa‑t. RNA synthetases) 4. Antibodies. Lysis with complement. Targets cell surface constituents mostly (e. g. , MHC) 5. Visual inspection at colony level: A. Sib selection (G 6 PD) 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 G 6 PD = 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- + Hprt-, TK+ Parental cells 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. , hn. RNP A 1 ), gene regulation (e. g. , turn on myogenesis) Synteny = genes physically linked on the same chromosome are syntenic. HAT medium HA T+ 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/~w 3 bio 315/lecture 2. htm
Complementation analysis Mutant parent 1 Mutant parent 2 gly 2 - + gly 1 - Mutant parent 1 Cell fusion + gly 1 - Hybrid cell gly. A- Glycine-free medium: No growth No complementation same gene (named gly. A) Mutant parent 2 gly 3 - Cell fusion Hybrid cell gly. A- gly. B- Glycine-free medium: Yes, growth Yes, complementation different genes (named gly. A and gly. B)
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)