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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 reach 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.

DNA transfection

DEAE= diethyl-amino-ethyl (positively charged)

DNA

DNA

polybrene

Linear PEI

Last updated: Nov. 21, 2011 1:10 AM

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Transient transfection vs. Permanent transfection: cloned genes chromosomally integratedunintegrated DNA. position effects ?unnatural ? (so analyze a pool of many tosuper-physiological expression average)levels (per transfected cell) ?

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 a high frequency of focus‑forming 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 for sensing growth factors

Mouse 3T3 cells Transformed Mouse 3T3 cells transfected with an EGFreceptor gene

Growth in soft agar

foci

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Lentivirus (a retrovirus)

RNA +strand genomes (2)

env

gag

pol

Viral transduction of genes

Also, adenovirus (DNA)

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Viral structural proteins contributed by packaging cell line(gag, env, pol, etc.)

DNA

Packaging signal on RNA

http://en.dogeno.us/2009/11/concentrate-retrovirus-carrying-vsv-g-envelope-by-ultracentrifugation/

Infects cells at high efficiency

Reverse transcribes to cDNA, integrates

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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 gancyclovirM. Capecchi, Nature Medicine  7, 1086 - 1090 (2001)

Generating mice with targeted mutations

Die in gancyclovir

Resistant to gancyclovir

ES cells and transgenic mice. Selection for 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 for human tk)

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Most K.O. work in ES cells mice homozygosis via F1 breedingLittle 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 factorES cells = embryonic stem cells

Gene knockouts via homologous recombination

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Chicken DT40 cells (high rate of homologous recombination

ASF

ASFASF

Human

+

ASFHuman

Tet-off promoter

ASF

ASF

neo

ASF

ASFHuman

hol

pur

neo

ASFHuman

pur+tetASF shut off

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. 1996,10:2588-99.

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

Select for HolR, Screen by Southern blotting

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Histidinol dehydrogenaseNAD+

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

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Gene amplification for high level production in CHO dhfr- cells.

DHFR system (dihydrofolate reductase): Selection for resistance to marginal levels of methotrexate

Folate DHFR

“FH2” “FH4”dihydrofolate tetrahydrofolate

DHFRGlycine

Purine nucleotides(AMP and GMP)

Thymidylic acid(TMP)

Methotrexate (MTX, amethopterin)

Resistance to MTX can occur via 3 different mechanaisms:1) Methotrexate permeation mutants (incl. MDR, increased efflux))2) Altered DHFR with lower MTX binding affinity3) Increased levels of P-glycoprotein efflux pump (MDR)4) Overproduction of DHFR protein

MDR

MDR = multiple drug resistance

11Gene 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, expanded chromosomal regions (HSRs)

HSRs are the location of the high number of dhfr genes.Double minute chromosomes are an occasional alternative form.

Amplicons (distance between repeated genes) are large (300 KB).(dhfr gene = ~ 25 kb)

HSRs can shrink, migrate.

12Reduction of folate to tetrahydrofolate

Reduction of folate to tetrahydrofolate

serine

glycine

FH4

FH2

CH2=FH4

FH4CH

PurBios

SHM

Dihydrofolate reductaseSerine hydroxym ethyltransferaseThym idylate synthetasePurine nucleotide biosynthetic pathw ayM ethylenetetrahydrofolate dehydrogenase

DHFR:SHM:TS:PurBios:M TDH:

TS

DHFR

dUMP

TMP

Precursors

IMP

Folate

DHFR

DNAMTDH

DNARNA

MTX

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Methotrexate:

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Tritium grains from hybridized cDNA

HSR:Homogenously staining region

Gene amplification

(R.T. Schimke, Sci. Amer. 243:60-69, 1980)

Nunberg et al. PNAS 1978 75:5553-6.

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“Homogeneously staining region”FISH, here

Gene amplification

FISH = fluorescent in situ hybridization

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HSR dmin upon DS break induced by a homing endonuclease (I-SceI).

HSR = homogeneously staining regionDmin = double minute chromosomes

Arnaud Coquelle, Lorène Rozier, Bernard Dutrillaux and Michelle Debatisse ONCOGENE, 31 October 2002, Volume 21, Number 50, Pages 7671-7679Induction of multiple double-strand breaks within an hsr by meganuclease I-SceI expression or fragile site activation leads to formation of double minutes and other chromosomal rearrangements

+FISH

Original locus?

Restriction-type enzyme with a very long recognition sequence ( ~20 bp)

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Ampification models: over-replication, unequal sister chromatid exchange, breakage and 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

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Gene amplification for 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 mediumMost 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) and thymdine

19DHFR- 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 and thymidine (GHT). In GHT-free medium CHO dhfr- cells die, but transfectants that have received a dhfr minigene, +/- YFG, survive.

X

serine

glycine

FH4

FH2

CH2=FH4

FH4CH

PurBios

SHM

Dihydrofo late reductaseSerine hydroxym ethyltransferaseThym idylate synthetasePurine nucleotide biosynthetic pathw ayM ethylenetetrahydrofolate dehydrogenase

DHFR:SHM:TS:PurBios:M TDH:

TS

DHFR

dUMP

TMP

Precursors

IMP

Folate

DHFR

DNAMTDH

DNARNA

DHFR-deficient cells require glycine, thymidine and a purine

TdR

hypoxanthine

3H-dUMP

3H-DNA

Xand are resistant to tritiated deoxyuridine

X

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Some other amplifiable genes

ENZYME ABBN INHIBITOR SELECTIVE MEDIUMAdenosine deaminase ADA deoxycoformycin MTX + adenosine

Ornithine decarboxylase ODC difluoromethyl-ornithine Polyamine-free

Asparagine synthetase AS Asparagine-free

Ribonucleoside reductase RR hydroxyurea Deoxynuceloside-free

Tri-functional pyrimidine synthetic enzyme

CAD PALA Pyrimidine-free

Thymidylate synthetase TS FUdR Thymidine-free

Dihydrofolate reductase DHFR MTX Gly-, TdR-, purine-free

Glutamine synthetase GS Methionine sulfoximine Gln-free

21A different major system for 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 for GS overproducers --->--> (gene amplification does not seem to be operating in this system of the GS cDNA gene and linked Mab genes)

Proprietary (Lonza Biologics)

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Transfection strategies

1. YFG (Your Favorite Gene) linked to a dhfr minigene on a single plasmidA. ~Insures co-integrationB. ~Insures co-amplification

2. YFG and dhfr on separate plasmidsA. Allows a high ratio of YFG to dhfr to startB. Co-amplification not assured but commonly occurs.

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Linked amp

dhfrYFG

Select in purine-free

m edium

DH FR+

D H FR-

DH FR+++++YFG +++++

Increas ing [M TX]

CHO cells

24Co-amp1

DHFR

DHFR YFG

YFG

Co-amplification of genes on unlinked plasmids

Transfection Co-integration, usually

Step-wiseLow MTX --> High MTXCo-am plification

W igler et al., PN AS, 1980 . 77: 3567-70 (Principle of co-amplification).

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DHFR YFG

YFG YFG

YFG

YFG

YFG

YFGYFG

YFG

YFG

YFG

YFG

YFG

YFG

YFG

YFG

Use a high ratio (e.g., 1000X) of YFG plasm id DNA to dhfr plasm id DNA

Transfect with 10 of and 10 of ug YFG ng dhfrCo-integration

(W igler et al, Cell, 1979: X174 DNA)

Multiple copies of from the startYFG

Step-w iseLow MTX --> High MTXCo-am plification

Co-amp3

(with or without pre-ligation)

76(11): 5684–5688

76: 5684–5688

26kaufman

Y.F.G.

Y.F.G.

DHFR

DHFRDicistronic mRNA

Transfection with both genes in one vector and even in one transcription unit

Poor translation initiationLow DHFRSupersensitive to MTXSelect initially in low MTXMore room for amplification

Kaufman, RJ, et al, NAR, 1993

(ribosome read-through)

Y.F.G. DHFRAlso, later, better dhfr translationusing an IRES,Internal ribosome initiation site, used mostly in viral but also in some cellular genes.In theory, not an advantage.

Y.F.G. DHFRDicistronic mRNA

IRES

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Co-amp2

DHFR

DHFR YFG

YFG

Use a weak dhfr promoter to confer supersensitivity to MTX

Transfection, Co-integration, usually

Stepwise low MTX --> high MTX

Co-amplificationVery

W igler et al., PN AS, 1980

More room for am plification

28Co-amp4

DHFR YFG

YFG YFG

YFG

YFG

YFG

YFGYFG

YFG

YFG

YFG

YFG

YFG

YFG

YFG

YFG

Use a high ratio (e.g., 1000X) of YFG plasm id DNA to dhfr plasmid DNAand a poorly expressed dhfr minigene

Transfect with 10 of and 10 of ug YFG ng dhfr

StepwiseVery low MTX --> high MTXCo-amplification

Co-integration

(W igler et al, Cell, 1979: X174 DNA)

Multiple copies of from the startYFG

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Pool of transfectants selected for growth in purine-free medium

Possible amplification protocol

0 10

10

40

20

20

80

40

40

160

80

80

300

160

1000

nM MTXin -GHT medium

nM MTX

nM MTX

etc. Finally clone several for final stages

Check selectedpopulations for Ig production

Amplification protocol

Note: Process is lengthy and tedious.

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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

31Top ten monoclonal antibodies in sales 2009-201