Transfection agents: CaPO4 (co-precipitates with DNA) Electroporation (naked DNA

Transfection agents: CaPO4 (co-precipitates with DNA) Electroporation (naked DNA

Transfection agents: CaPO4 (co-precipitates with DNA) Electroporation (naked DNA

Borasio, James, Morning Drive On-Air Personality has reference to this Academic Journal, PHwiki organized this Journal Transfection agents: CaPO4 (co-precipitates with DNA) Electroporation (naked DNA, high voltage pulse transient holes) Lipofection (multilamellar liposomes) Polybrene (detergent) Ballistic (DNA-coated gold particles) DEAE-dextran (toxic, OK as long as transient) Poly-ethylenimine (PEI, cheap) Effectene (non-liposomal lipid) Must traverse cytoplasm. Much engulfed in lysosomes. Inhibition of lysosomal function often helps (chloroquin). Co-integration of high MW DNA . Can reach 2000 KB. Separate plasmids transfected together same site (co-integration). Separate transfections separate locations R in addition to om or semir in addition to om (many) integration sites (unless targeted) Low but real homologous recombination rate. DNA transfection DEAE= diethyl-amino-ethyl (positively charged) Last updated: Nov. 21, 2011 1:10 AM Transient transfection vs. Permanent transfection: cloned genes chromosomally integrated unintegrated DNA. position effects unnatural (so analyze a pool of many to super-physiological expression average) levels (per transfected cell) Transient -> 1090% transfection efficiency (stain) Permanents more like 0.001 transfectants per g DNA per cell (~high). i.e., 106 treated cells -> 1000 colonies; could be much less as long as certain types of cells One the most dramatic first applications of gene transfection from total DNA: Transfer of the growthtrans as long as med 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 as long as foci (one = focus). Make a library from growthtrans as long as med transfectant. Screen as long as human Alu repeat. Verify that cloned DNA yields a high frequency of focus as long as ming transfectants. Isolate cDNA by hybridization to the cloned genomic DNA. Sequence. Identify gene/protein: = a dominant oncogene. Ras, a signaling protein in a transducing pathway as long as sensing growth factors Mouse 3T3 cells Trans as long as med Mouse 3T3 cells transfected with an EGFreceptor gene Growth in soft agar foci

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Lentivirus (a retrovirus) RNA +str in addition to genomes (2) env gag pol Viral transduction of genes Also, adenovirus (DNA) Viral structural proteins contributed by packaging cell line (gag, env, pol, etc.) DNA Packaging signal on RNA Infects cells at high efficiency Reverse transcribes to cDNA, integrates HSV-TK gene is removed during homologous recombination, but remains 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 M. Capecchi, Nature Medicine 7, 1086 – 1090 (2001) Generating mice with targeted mutations Die in gancyclovir Resistant to gancyclovir ES cells in addition to transgenic mice. Selection as long as homologous recombinants via the loss of HSV TK genes (Capecchi): –– hsvtk – homol. region – drugR – homol. region – hsvtk –– Non-homologous recombination favors ends: tk is inserted, conferring sensitivity to the drug gancyclovir (HSVtk specific, not a substrate as long as human tk)

Most K.O. work in ES cells mice homozygosis via F1 breeding Little work in cultured lines: Myc double sequential K.O. = viable, ~sick (J. Sedivy, Genes & Dev. 1998. 12: 3797-3802 ) Splicing factor (ASF) double K.O. see next graphic. ASF = alternative splicing factor ES cells = embryonic stem cells Gene knockouts via homologous recombination Chicken DT40 cells (high rate of homologous recombination + Tet-off promoter hol pur neo pur +tet ASF shut off cell viable (covered by human ASF gene neo X pur Cell dies without ASF (follow events biochemically) ASF- ASF- ASF- Double knockout of the ASF gene, a vital gene, by homologous recombination Wang, Takagaki, in addition to Manley, Targeted disruption of an essential vertebrate gene: ASF/SF2 is required as long as cell viability. Genes Dev. 1996,10:2588-99. neo neo Hol = histidinol resistance; pur = puromycin resistance Drug resistance genes here chosen as long as illustration. hol One ASF gene allele disrupted by homologous recombination Both alleles have been disrupted in some purR, holR cells Select as long as HolR, Screen by Southern blotting protein synthesis inhibits protein synthesis (competitive inhibitor of histidinyl-tRNA synthetase) Protein synthesis stops at a histidine codon Histidinol dehydrogenase detoxifies histidinol, confers histidinol resistance

Gene amplification as long as high level production in CHO dhfr- cells. DHFR system (dihydrofolate reductase): Selection as long as resistance to marginal levels of methotrexate Folate DHFR “FH2” “FH4” dihydrofolate tetrahydrofolate DHFR Glycine Purine nucleotides (AMP in addition to GMP) Thymidylic acid (TMP) Resistance to MTX can occur via 3 different mechanaisms: 1) Methotrexate permeation mutants (incl. MDR, increased efflux)) 2) Altered DHFR with lower MTX binding affinity 3) Increased levels of P-glycoprotein efflux pump (MDR) 4) Overproduction of DHFR protein MDR MDR = multiple drug resistance Gene amplification: dhfr Historically: Methotrexate resistance MTX inhibits dihydrofolate reductase (DHFR) MTX-resistant cells have (in order of discovery): High DHFR enzyme activity High DHFR protein High protein synthetic rate High in vitro translatable mRNA High mRNA level (by hybridization) High DNA level. Homogeneously staining, exp in addition to ed chromosomal regions (HSRs) HSRs are the location of the high number of dhfr genes. Double minute chromosomes are an occasional alternative as long as m. Amplicons (distance between repeated genes) are large (300 KB). (dhfr gene = ~ 25 kb) HSRs can shrink, migrate. Reduction of folate to tetrahydrofolate MTX

Methotrexate: HSR: Homogenously staining region Gene amplification (R.T. Schimke, Sci. Amer. 243:60-69, 1980) Nunberg et al. PNAS 1978 75:5553-6. “Homogeneously staining region” FISH, here Gene amplification FISH = fluorescent in situ hybridization

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 in addition to Michelle Debatisse ONCOGENE, 31 October 2002, Volume 21, Number 50, Pages 7671-7679 Induction of multiple double-str in addition to breaks within an hsr by meganuclease I-SceI expression or fragile site activation leads to as long as mation of double minutes in addition to other chromosomal rearrangements Original locus Restriction-type enzyme with a very long recognition sequence ( ~20 bp) Ampification models: over-replication, unequal sister chromatid exchange, breakage in addition to fusion (Tanaka et al. Mol. Cell. Biol. 2007 27:1993-2002 .). Map dhfr amplicons: ~ 300 kb , but wide range (Nunberg et al., Proc Natl Acad Sci U S A. 1978 Nov;75(11):5553-6; Looney et al., Mol Cell Biol. 1988. 8:5268-79) Gene amplification is rare in normal cells p53- mutation allows it. (Livingstone et al., Cell. 1992.70:923-35; Yin et al., Cell. 1992. 70:937-48). 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 MDR = multiple drug resistance Gene amplification as long as 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. -GHT = without glycine, hypoxanthine (a purine source) in addition to thymdine

DHFR- cells require G,H,T DHFR- cells selected by their resistance to radioactive 3H-deoxyuridine: 3H-dU 3H-dUMP 3H-TMP 3H-DNA death from radioactive decay. DHFR- cells require glycine, hypoxanthine in addition to thymidine (GHT). In GHT-free medium CHO dhfr- cells die, but transfectants that have received a dhfr minigene, +/- YFG, survive. X in addition to are resistant to tritiated deoxyuridine X Some other amplifiable genes A different major system as long as 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 as long as GS overproducers –>-> (gene amplification does not seem to be operating in this system of the GS cDNA gene in addition to linked Mab genes) Proprietary (Lonza Biologics)

Transfection strategies YFG (Your Favorite Gene) linked to a dhfr minigene on a single plasmid A. ~Insures co-integration B. ~Insures co-amplification YFG in addition to dhfr on separate plasmids A. Allows a high ratio of YFG to dhfr to start B. Co-amplification not assured but commonly occurs. Linked amp CHO cells Co-amp1 . 77: 3567-70 (Principle of co-amplification).

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Co-amp3 (with or without pre-ligation) 76(11): 5684–5688 76: 5684–5688 kaufman (ribosome read-through) Y.F.G. DHFR Also, later, better dhfr translation using an IRES, Internal ribosome initiation site, used mostly in viral but also in some cellular genes. In theory, not an advantage. Y.F.G. DHFR Dicistronic mRNA IRES Co-amp2

Co-amp4 Amplification protocol Note: Process is lengthy in addition to tedious. Some marketed recombinant proteins Erythropoietin (Epogen, Procrit) J&J, Amgen Tissue plasminogen activator (TPA) Genentech Growth Hormone (Genentech) Insulin (Genentech) Beta-interferon (Avonex) Biogen-IDEC Alpha-interferon (IntronA) Schering-Plough Neupogen (Amgen) Etanercept – TNF receptor + IgG (Enbrel) Amgen Monoclonal antibodies (mAbs): see next graphic

Top ten monoclonal antibodies in sales 2009-201

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