1. 1 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 Last updated Nov. 16, 12:10 AM

2. 2 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 (previously discussed) 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 )

3. 3 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 Chromatin organization: e.g., histone acetylation (active) / de-acetylation (inactive) 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)

4. 4 Solutions to diploidy problem: Double mutants: heavy mutagenesis, mutants/survivor increases but mutants/ml decreases Incl. also mutation + segregation, or mutation + homozygosis: (rare but does occur) 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 0.0007 2 ~ 5X10 -7. One 10cm tissue culture dish holds ~ 10 7 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 and sequence 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

5. 5 + - + - + - + - - + + - or Heterozygote After homologous recombination (not sister chromatid exchange) - - + - + 2 heterozygotes again 1 homozygote +/+ 1 homozygote -/- Homozygosis: Loss of heterozygosity (LOH) by mitotic recombination between homologous chromosomes (rare) Paternal Chr. 4, say Maternal Chr. 4 Recombinant chromatids L R L R L R R L L L R R Recessive phenotype is unmasked = a mechanism of homozygosis of recessive tumor suppressor mutations in cancer MitosisMitosis

6. 6 Mutagenesis (induced general mutations, not site directed) Chemical and physical agents: MNNGpoint mutations (single base substitutions) EMS point mutations (single base substitutions) Bleomycin small deletions UVmostly 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. MNNG = methyl N-nitrosoguanidine EMS = ethylmethane sulfonate

7. 7 Categories of cell mutant selections Example Auxotrophs(via BrdU selection)purine-; pyrimidine-; glyc-, pro-;gln- Drug resistance Dominant ouabain R, alpha-amanitin R Recessive6-TG r, BrdU r Antibodies vs. surface componentsMHC- Visual inspectionG6PD-, Ig IP- FACS = fluorescence ‑ activated cell sorterDHFR- Brute force screening IgG-, electrophoretic shifts Temperature ‑ sensitive mutants 3 H-leu resistant (leucyl tRNA synthetase-) TG = 6-thioguanine; BrdU = 5-bromodeoxyuridine; MHC = majpor histocompatibility locus; G6PD = glucose-6-phosphate dehydrogenase

8. 8 Auxotroph selection by killing growing cells: Mutant cell cannot grow in deficient medium so does not incorporate BrdU (BUdR) and so survives DNA damage from subsequent treatment with 313 nm light Kao and Puck, PNAS

9. 9 (drugs, in italics) PRPP = phosphoribosyl pyrophosphate; FH 4 =tetrahydrofolate DHFR

10. 10 Methotrexate (=amethopterin) (~aminopterin) Folate Glycine Thymidine (T) FH4 PRPP + glutamine IMP Adenylosucc. AMP Nuc. Acid GMPXMP HGPRT XGPRT (Eco gpt) Nuc. Acid APRT Adenosine kinase Adenosine Adenine(A) (diaminopurine, DAP) (8-azaadenine, 8AA) Hypoxanthine (H) Guanine (6-thioguanine, 6TG) (8-azaguanine, 8AG) Xanthine (X) Azaserine Glutamine Purine biosynthesis, salvage pathways, and inhibitors Alanosine Mycophenolic in italics Only mutation +GHT-GHT-GHT +6TG -GHT + DAP -H +GT +MTX + A -H +GT +MTX + Guanine -H +GT +MTX + H WT APRT- HPRT- DHFR- 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 APRT = adenine phosphoribosyltransferase Growth pattern examples + Test yourself: Fill in the boxes Grow (+) or not grow(-) Click here for the answershere - + -

11. 11 1. Auxotrophs (BrdU reverse selection) 2. Drug resistance (dominants or recessives) 3. Temperature ‑ sensitive mutants: cell cycle mutants. Tritiated amino acid suicide (aa ‑ tRNA synthetases) 4. Antibody resistance. 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 mutant types:

12. 12 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)

13. 13 PEG (polyethylene glycol, mw ~ 6000 Sendai virus, inactivated + Cell fusion Parental cells Heterokaryon (alternative = a homokaryon) Cell cycle, Nuclear fusion, Mitosis, Survival, reproducton Hybrid cell 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 (dominance/recessiveness) Complementation HAT medium Hprt-, TK+ Hprt+, TK- Hprt-, TK+ Hprt+ TK- HAT- HAT+ Hprt-, TK+, Hprt+ TK- Cell fusion Synteny = genes physically linked on the same chromosome are syntenic.

14. 14 Frye and Edidin, 1970: Use of cell fusion and heterokaryons to measure the diffusion of membrane proteins http://www.erin.utoronto.ca/~w3bio315/lecture2.htm Complete mixing in < 40 min. No diffusion at low temperature (<15-20 deg) t=0 t=40’

15. 15 + Cell fusion Hybrid cell Glycine-free medium: No growth No complementation  same gene (named glyA) gly1- glyA- gly2- Complementation analysis + Cell fusion Hybrid cell gly1- glyA- glyB- gly3- Glycine-free medium: Yes, growth Yes, complementation  different genes genes (named glyA and glyB) Mutant parent 1 Mutant parent 2Mutant parent 1 Mutant parent 2

16. 16 Nuclear-cytoplasmic shuttling demonstrated using interspecific heterokaryons Fused cell: HeLa + frog Unfused frog cells Frog nuclei in fused cell CHX = cycloheximide (protein synthesis inhibitor) given 0.5 h before fusion HnRNP C HnRNP A1 A1 shuttles, C does not. Pinal-Roma and Dreyfuss, Nature, 355:730

17. 17 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 for 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 = 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 (at P&S: Wigler, Axel and Silverstein) DNA transfection DEAE= diethyl-amino-ethyl (positively charged) DNA polybrene Linear PEI

18. 18 Mike Wigler Richard Axel Saul Silverstein History: discovered for practical use at Columbia (P&S: Wigler Axel and Silverstein)

19. 19 Transient transfection vs. permanent: cloned genes Unintegrated DNA chromosomally integrated. Unnatural? Position effects ? Super-physiological expression (so analyze a pool of many to levels (per transfected cell) ? average) Transient -> 10 ‑ 90% transfection efficiency (stain) Permanents more like 0.001 transfectants per μg DNA per cell (~high). i.e., 10 6 treated cells -> 1000 colonies; could be much less for certain types of cells

20. 20 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 that cloned DNA yields high frequency of focus ‑ forming transfectants. Isolate cDNA by hybridization to the cloned genomic DNA. Sequence. Identify gene: = a dominant oncogene. Ras, a signaling protein in a transducing pathway for sensing growth factors Mouse 3T3 cells Transformed Mouse 3T3 cells transfected with an EGFreceptor gene

21. 21 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.

22. 22 ES cells and transgenic mice. Selection for homologous recombinants via the loss of HSV TK genes (Capecchi): – tk – homol. region – drug R – homol. region – tk – Non-homologous recombination favors ends; tk is inserted, conferring sensitivity to the drug gancyclovir (HSVtk specific, not a substrate for human tk) 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. see next graphic. APRT = adenine phosphoribosyltransferase ASF = alternative splicing factor Gene knockouts via homologous recombination

23. 23 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, today M. Capecchi, Nature Medicine 7, 1086 - 1090 (2001) Generating mice with targeted mutations Die in gancyclovir Resistant to gancyclovir

24. 24 Chicken DT40 cells ASF Human + ASF Human Tet-off promoter ASF neo ASF Human hol pur neo ASF Human pur +tet cell viable (covered by human ASF gene neo ASF Human X pur Cell dies without ASF (follow events biochemically) ASF- Double knockout of the ASF gene, a vital gene, by homologous recombination Wang, Takagaki, and Manley, Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability. Genes Dev. 1996 Oct 15;10(20):2588-99. neo Hol = histidinol resistance; pur = puromycin resistance Drug resistance genes here chosen for illustration. hol One ASF gene allele disrupted by homologous recombination Both alleles have been disrupted in some purR, holR cells

25. 25 Histidinol dehydrogenaseNAD + protein synthesis inhibits protein synthesis (charged to tRNA but cannot be transferred to growing peptide so truncates) Histidinol dehydrogenase detoxifies histidinol, confers histidinol resistance