Mammalian cell genetics
Last updated Nov. 16, 12:10 AM
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
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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)
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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 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)
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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 0.00072~ 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
Homozygosis:
Loss of heterozygosity (LOH)
by mitotic recombination between
homologous chromosomes (rare)
L
R
L
M
i
t
o
s
i
s
R
5
L
R
--
R
L
+
-
+
2 heterozygotes again
L
R
L
R
or
+
+
-
-
Paternal
Maternal
Chr. 4, say Chr. 4
Heterozygote
+
-
+
-
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
-
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Mutagenesis (induced general mutations, not site directed)
Chemical and physical agents:
MNNG
EMS
Bleomycin
UV
Ionizing radiation (X-, gamma-rays)
point mutations (single base substitutions)
point mutations (single base substitutions)
small deletions
mostly point mutations but also large deletions
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
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Categories of cell mutant selections
•
Auxotrophs (via BrdU selection)
Example
purine-; pyrimidine-;
glyc-, pro-;gln-
•
Drug resistance
Dominant
Recessive
ouabainR, alpha-amanitinR
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
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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
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Purine biosynthesis, salvage pathways, and inhibitors
Adenine(A)
(diaminopurine)
(8-azaadenine)
Methotrexate
(=amethopterin)
(~aminopterin)
Folate
Adenosine
DHFR
APRT
Adenosine
kinase
FH4
AMP
Nuc. Acid
GMP
Nuc. Acid
Glycine
Thymidine (T)
Alanosine Adenylosucc.
PRPP +
glutamine
Azaserine
Glutamine
IMP
HGPRT
XMP
Hypoxanthine
(H)
XGPRT
(Eco gpt)
Xanthine
(X)
Mycophenolic
acid
Code:
Biosynthesis;
Salvage enzymes
Inhibitors
(drugs, in italics)
Analogs (iytal.)
PRPP = phosphoribosyl pyrophosphate; FH4=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, 8AA)
Methotrexate
(=amethopterin) Folate
(~aminopterin)
Adenosine
APRT
Adenosine
FH4
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
-GHT
+6TG
IMP
HGPRT
XMP
Hypoxanthine XGPRT
(H)
(Eco gpt)
-GHT +
DAP
Mycophenolic Xanthine
(X)
-H +GT
+MTX
-
GMP
Nuc. Acid
HGPRT
Guanine
(6-thioguanine, 6TG)
(8-azaguanine, 8AG)
-H +GT
-H +GT
in italics
+MTX
+MTX +
Guanine
+A
+
-
-H +GT
+MTX
+H
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Cell mutant types:
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
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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 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)
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Cell fusion
Hprt+, TK-
+
Parental cells
Hprt-, TK+
HAT-
HAT-
PEG (polyethylene glycol, mw ~ 6000
Sendai virus, inactivated
Cell fusion
Hprt-, TK+
Heterokaryon
(alternative = a homokaryon)
Hybrid cell
Hprt+ TK-
Heterokaryon use examples:
membrane dynamics (lateral diffusion of
membrane proteins)
shuttling proteins (e.g., hnRNP A1 ),
gene regulation (e.g., turn on myogenesis)
Synteny = genes physically linked on
the same chromosome are syntenic.
HAT
medium
HAT+
Cell cycle,
Nuclear fusion,
Mitosis,
Survival,
reproducton
Hprt-, TK+,
Hprt+ TK-
Hybrid cells: examples of use:
gene mapping (synteny)
gene regulation (dominance/recessiveness)
Complementation
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Frye and Edidin, 1970:
Use of cell fusion and heterokaryons to measure the diffusion of membrane
proteins
t=0
t=40’
Complete mixing in < 40 min.
No diffusion at low temperature (<15-20 deg)
http://www.erin.utoronto.ca/~w3bio315/lecture2.htm
Mutant parent 2
Mutant parent 1
gly2-
+
gly1-
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Complementation analysis
Mutant parent 1
Cell fusion
+
Glycine-free medium: No growth
No complementation
same gene
(named glyA)
gly3-
gly1Cell fusion
Hybrid cell
glyA- glyA-
Mutant parent 2
Hybrid cell
glyA- glyB-
Glycine-free medium: Yes, growth
Yes, complementation
different genes genes
(named glyA and glyB)
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Nuclear-cytoplasmic shuttling demonstrated using interspecific heterokaryons
HnRNP A1
HnRNP C
Unfused frog cells
Fused cell: HeLa + frog
A1 shuttles, C does not.
Frog nuclei in fused cell
Pinal-Roma and Dreyfuss,
Nature, 355:730
CHX = cycloheximide (protein synthesis inhibitor) given 0.5 h before fusion
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DNA transfection
Transfection agents:
CaPO4 (co-precipitates with DNA)
Electroporation (naked DNA, high voltage pulse transient holes)
Lipofection (multilamellar liposomes)
Polybrene (detergent)
DNA
Ballistic (DNA-coated gold particles)
DEAE-dextran (toxic, OK for transient)
polybrene
Poly-ethylenimine (PEI, cheap)
Effectene (non-liposomal lipid)
DNA
Must traverse cytoplasm. Much engulfed in lysosomes.
Inhibition of lysosomal function often helps (chloroquin).
Linear PEI
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)
DEAE= diethyl-amino-ethyl (positively charged)
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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.
Unintegrated DNA
Unnatural?
Super-physiological expression
levels (per transfected cell) ?
permanent: cloned genes
chromosomally integrated.
Position effects ?
(so analyze a pool of many to
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.
Gene knockouts via homologous recombination
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 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
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Resistant to
gancyclovir
HSV-TK gene is removed during
homologous recombination, but
remains joined during nonhomologous recombination.
Unlike mammalian TK, HSVTk
converts gancyclovir to a toxic
product
Die in
gancyclovir
M. Capecchi, Nature Medicine 7, 1086 - 1090 (2001)
Generating mice with targeted mutations
HSV = Herpes simplex virus; tk =
thymidine kinase; FIAU = equivalent
to gancyclovir, today
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Double knockout of the ASF gene, a vital gene, by homologous recombination
Chicken DT40 cells
+
Human
ASF
neo
ASF
ASF
ASF
One ASF gene
allele disrupted by
homologous
recombination
hol
neo
hol
ASF
ASF-
ASF
Human
ASF
Human
ASF
neo
Tet-off promoter
Hol = histidinol resistance; pur = puromycin resistance
Drug resistance genes here chosen for illustration.
neo
pur
Human
X ASF
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.
Cell dies without ASF
(follow events biochemically)
pur
Both alleles have been
disrupted in some
purR, holR cells
neo
+tet
pur
ASFASF-
Human
ASF
cell viable
(covered by human ASF gene
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Histidinol dehydrogenase detoxifies histidinol, confers histidinol resistance
protein synthesis
NAD+
Histidinol dehydrogenase
inhibits protein synthesis
(charged to tRNA but cannot be
transferred to growing peptide so truncates)