Transfecting DNA into mammalian cells

1
Transfecting DNA into mammalian cells
Donor
Target
Elsewhere in the genome
HR
NHEJ
Targeted gene replacement
Random integration
2
Targeting ratio as a function of delivery method
in mammalian cells
Transfection method
Transfection efficiency
Random integration
Gene targeting
Targeting ratio
Microinjection
ND
1.2 x 10-1
8 x 10-3
115
Electroporation
8.7 x 10-4
10
2.1 x 10-7
12,400
CaPhosphate
2.5 x 10-2
24
6.3 x 10-7
140,000
Fugene-6
4.8 x 10-2
92
1.3 x 10-7
1370,000
Lipofectamine
2.2 x 10-1
98
6.2 x 10-7
1350,000
Vasquez et al. (2001) PNAS 98 8403
3
Two modes of homologous gene targeting
Ends-in
Ends-out
Donor
Target
Insertion
Replacement
4
Gene replacement in yeast
Trp1 gene
Gene of interest (in Trp1- strain)
PCR with primers having 5 extensions
corresponding to flanks of gene of interest
Homologous recombination
Trp1 replaces gene of interest. Select on -Trp
medium
5
Positive-Negative (PN) Selection
neoR
TK
TK
Targeting vector
Gene to target
Nonhomologous integration
Homologous recombination
neoR TK- G418R gancR
neoR TK G418R gancS
6
From Lufkin (1995) in Gene Targeting, M.A. Vega,
ed., CRC Press, p. 123
7
Conditional Gene Targeting in Mice
Haldar et al. Capecchi (2007) Cancer Cell 11
375
8
And verify by Southern blot
From Nicholl (2002) An Introduction to Genetic
Engineering, 2nd ed., Cambridge Univ. Press
9
From Nicholl (2002) An Introduction to Genetic
Engineering, 2nd ed., Cambridge Univ. Press
10
P element-mediated transgenesis in Drosophila
P element ends
Inject into embryos Transposase
expressed Transposition of GOI activated Insertion
into genome at random
Gene of interest
P transposase
11
Basic retrovirus structure
LTR
LTR
gag
pol
env
Y
Retroviral vector
LTR
LTR
PCMV
Transgene
Y
Helper cell
gag
pol
env
PCMV
polyA
polyA
PCMV
12
Gene Therapy of Human Severe Combined
Immunodeficiency (SCID)-X1 Disease Marina
Cavazzana-Calvo, 123 Salima Hacein-Bey, 123
Geneviève de Saint Basile, 1 Fabian Gross, 2 Eric
Yvon, 3 Patrick Nusbaum, 2 Françoise Selz, 1
Christophe Hue, 12 Stéphanie Certain, 1
Jean-Laurent Casanova, 14 Philippe Bousso, 5
Françoise Le Deist, 1 Alain Fischer 124 Severe
combined immunodeficiency-X1 (SCID-X1) is an
X-linked inherited disorder characterized by an
early block in T and natural killer (NK)
lymphocyte differentiation. This block is caused
by mutations of the gene encoding the c cytokine
receptor subunit of interleukin-2, -4, -7, -9,
and -15 receptors, which participates in the
delivery of growth, survival, and differentiation
signals to early lymphoid progenitors. After
preclinical studies, a gene therapy trial for
SCID-X1 was initiated, based on the use of
complementary DNA containing a defective c
Moloney retrovirus-derived vector and ex vivo
infection of CD34 cells. After a 10-month
follow-up period, c transgene-expressing T and NK
cells were detected in two patients. T, B, and NK
cell counts and function, including
antigen-specific responses, were comparable to
those of age-matched controls. Thus, gene therapy
was able to provide full correction of disease
phenotype and, hence, clinical benefit
M. Cavazzana-Calvo et al., Science 288,
669-672 (2000)
13
Retroviral vector for expression of ?c in human
cells
LTR
LTR
IL-2R?
SD
SA
?
mRNA
Procedure Harvest bone marrow Collect CD34
cells Infect with viral vector on 3 consecutive
days (30 of cells expressed ?c) Infuse into
patients
14
M. Cavazzana-Calvo et al., Science 288,
669-672 (2000)
Published by AAAS
15
Hacien-Bey-Abina (2002) NEJM 346 1185
16
Fig. 1. Kinetics and characteristics of P4 and P5
abnormal T cells
Treatments for T-cell leukemia
S. Hacein-Bey-Abina et al., Science 302,
415-419 (2003)
Published by AAAS
17
Fig. 3. LMO2 expression in clonal T cells
S. Hacein-Bey-Abina et al., Science 302,
415-419 (2003)
Published by AAAS
18
Characteristics of PhiC31 Integrase
Groth Calos (2004) J. Mol. Biol. 335 667
19
Thyagarajan et al. (2001) Mol. Cell. Biol. 21
3926
20
TABLE 1. Integration frequency at native and
inserted attP sites
-------------- G418r integrants------------------
----- (-) Int (n) () Int
(n) Fold increase Human cells
293 0.008 0.0001 (172) 0.069 0.002 (1,306)
8.6 293P1 0.013 0.0007 (20) 0.18 0.01
(190) 13.8 293P2 0.013 0.003 (17) 0.15
0.04 (191) 11.5 293P3 0.02 0.002 (48) 0.25
0.02 (386) 12.5 293P4 0.014 0.002 (21) 0.24
0.01 (269) 17.1 Mouse cells 3T3 0.25
0.005 (136) 1.2 0.2 (253) 4.8 3T3P1 0.27
0.04 (191) 2.6 0.5 (331) 9.5 3T3P2 0.19
0.03 (162) 1.7 0.3 (428) 9.1 3T3P3 0.34
0.01 (74) 4.6 1 (476) 13.5
Cells labeled P1, P2, etc. have synthetic attP
sites in their genomes, and these are used
approximately 15 of the time. Other lines (293,
3T3) use only pseudo-attP sites.
Thyagarajan et al. (2001) Mol. Cell. Biol. 21
3926
21
Delivery of a human Factor IX gene to mice with
PhiC31 integrase by tail vein injection
Olivares et al. (2002) Nature Biotechnol. 20 1124
22
Donor
Target
10-5-10-6
Product
23
Zinc finger nucleases (ZFNs)
Each finger contacts three consecutive base pairs
in the target DNA. New sequence specificities can
be created by altering a few specific residues in
the protein.
Kim, Cha Chandrasegaran (1996) PNAS 93 1156
24
Zinc finger nucleases (ZFNs)
3
2
1
FN
5
gt
3
FN
3
2
1
The cleavage domain must dimerize to cut
DNA. Cleavage leaves a 4-nt 5 overhang. There
are zinc fingers available for many triplets.
25
Targeting Drosophila rosy
Xba I
TGA TAA

TCTAGA
I-SceI
I-SceI


AGC TAC TAC TCG ATG ATG
acgaat tgctta
GGC GTG GGA CCG CAC CCT
26
ryA
ryB
ry
ry
NHEJ
ry
ry-
27
High efficiency targeting at ry
with Mutants Parents Mutants per Parent
HR Female 174 95 17.5 59 Male
148 90 11.1 36
NHEJ mutations are mostly small deletions and
insertions, sometimes larger deletions up to
several hundred bp.
28
Mutations in the ?c gene
Urnov et al. (2005) Nature 435 646
29
Urnov et al. (2005) Nature 435 646
30
RNA interference (RNAi)
Zamore (2002) Science 296 1265.
31
Zamore (2002) Science 296 1265.
32
Plasterk (2002) Science 296 1263.