1 Mammalian cell geneticsIntroduction:

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

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Mammalian cell geneticsAdvantages of cultured cells (vs. whole organism): numbers, homogeneity

Disadvantages of cultured mammalian cells: limited phenotypeslimited 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)

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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 don’t yet understand this).

DNA CpG methylationChromatin 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)

4Solutions 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 0.00072~ 5X10-7. One 10cm tissue culture dish holds ~ 107 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

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+ -+ -+ - + -

-+ + -

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 RL R

LR RL

L L R R

Recessive phenotype is unmasked

= a mechanism of homozygosis of recessive tumor suppressor mutations in cancer

Mitosis

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Mutagenesis (induced general mutations, not site directed)

Chemical and physical agents: MNNG point mutations (single base substitutions)EMS point mutations (single base substitutions)Bleomycin small deletionsUV 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.

MNNG = methyl N-nitrosoguanidineEMS = ethylmethane sulfonate

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Categories of cell mutant selections

Example• Auxotrophs (via BrdU selection) purine-; pyrimidine-; glyc-, pro-;gln-• Drug resistance

Dominant ouabainR, alpha-amanitinR

Recessive 6-TGr, BrdUr

• Antibodies vs. surface components MHC-

• Visual inspection G6PD-, Ig IP-

• FACS = fluorescence‑activated cell sorter DHFR-

• Brute force screening IgG-, electrophoretic shifts

• Temperature‑sensitive mutants 3H-leu resistant (leucyl tRNA synthetase-)

TG = 6-thioguanine; BrdU = 5-bromodeoxyuridine; MHC = majpor histocompatibility locus; G6PD = glucose-6-phosphate dehydrogenase

8Auxotroph selection by killing growing cells:

Mutant cell cannot grow in deficient mediumso does not incorporate BrdU (BUdR) and so survives DNA damage from subsequent treatment with 313 nm light

Kao and Puck, PNAS

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Methotrexate(=amethopterin)(~aminopterin)

Folate

GlycineThymidine (T)

FH4

PRPP + glutamine

IMP

Adenylosucc.

AMP Nuc. Acid

GMPXMPHGPRT

HGPRTXGPRT(Eco gpt)

Nuc. Acid

APRTAdenosine kinase

Adenosine

Adenine(A) (diaminopurine)(8-azaadenine)

Hypoxanthine(H)

Guanine(6-thioguanine)(8-azaguanine)

Xanthine(X)

Azaserine

Glutamine

Purine biosynthesis, salvage pathways, and inhibitors

Alanosine

Mycophenolicacid

Code: Biosynthesis; Salvage enzymes Inhibitors Analogs (iytal.)(drugs, in italics)

PRPP = phosphoribosyl pyrophosphate; FH4=tetrahydrofolate

DHFR

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Methotrexate(=amethopterin)(~aminopterin)

Folate

GlycineThymidine (T)

FH4

PRPP + glutamine

IMP

Adenylosucc.

AMP Nuc. Acid

GMPXMPHGPRT

HGPRTXGPRT(Eco gpt)

Nuc. Acid

APRTAdenosine 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 italicsOnly 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-

GHT = glycine, hypoxanthine, and thymidineA = adenine H = hypoxanthineG = glycineTG = 6-thioguanine (G analog) DAP = diaminopurine (A analog)MTX = methotrexate (DHFR inhibitor)DHFR = dihydrofolate reductaseHPRT = hypoxanthine-guanine phosphoribosyltransferaseAPRT = adenine phosphoribosyltransferase

Growth pattern examples

+

Test yourself: Fill in the boxesGrow (+) or not grow(-)Click here for the answers

-

+ -

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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 proteinsG6PD = glucose-6-phosphate dehydrogenase; LCH = lactate dehydrogenase; Ig = immunoglobulin. IP = immunoprecipitate

Cell mutant types:

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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 selectionDominance 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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PEG (polyethylene glycol, mw ~ 6000Sendai virus, inactivated

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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-

HAT+

Hprt-, TK+,Hprt+ TK-

Cell fusion

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 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’

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+

Cell fusion

Hybrid cell

Glycine-free medium: No growth No complementationsame gene (named glyA)

gly1-

glyA- glyA-

gly2-

Complementation analysis

+

Cell fusion

Hybrid cell

gly1-

glyA- glyB-

gly3-

Glycine-free medium: Yes, growth Yes, complementationdifferent genes genes (named glyA and glyB)

Mutant parent 1Mutant parent 2 Mutant parent 1 Mutant parent 2

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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 CHnRNP A1

A1 shuttles, C does not.

Pinal-Roma and Dreyfuss, Nature, 355:730

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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 locationsRandom 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

DNA

polybrene

Linear PEI

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Mike Wigler Richard Axel Saul Silverstein

History: discovered for practical use at Columbia (P&S: Wigler Axel and Silverstein)

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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., 106 treated cells -> 1000 colonies; could be much less for certain types of cells

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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

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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.

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ES cells and transgenic mice. Selection for homologous recombinants via the loss of HSV TK genes (Capecchi): – tk – homol. region – drugR – 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 breedingLittle work in cultured lines:Myc double sequential K.O. = viable, ~sick (J. Sedivy)Splicing factor (ASF) double K.O. see next graphic.APRT = adenine phosphoribosyltransferaseASF = alternative splicing factor

Gene knockouts via homologous recombination

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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

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Chicken DT40 cells

ASF

ASFASF

Human

+

ASFHuman

Tet-off promoter

ASF

ASF neo

ASF

ASFHuman

hol

pur

neo

ASFHuman

pur+tet

cell viable(covered by human ASF gene

neo

ASFHuman

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, 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

neo

Hol = histidinol resistance; pur = puromycin resistanceDrug 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

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Histidinol dehydrogenaseNAD+

protein synthesis

inhibits protein synthesis(charged to tRNA but cannot betransferred to growing peptide so truncates)

Histidinol dehydrogenase detoxifies histidinol, confers histidinol resistance