On the All or Half Law of Recombinant DNA, Lentivus Transduction and some others.pptx - Copy
1. On the “All or Half” law of Recombinant
DNA, Lentivirus Transduction and Some
Others
Gang Zhang, Ph. D
Research Technician II
Centre for Research in Neurodegenerative Diseases
Department of Medicine
University of Toronto, Canada
2. Main Research Experience
2001-2005: Ph. D. The School of Life Sciences, Shandong Normal University (2001-2002),
Institute of Zoology, Chinese Academy of Sciences (2002-2005). Project: Mouse cloning and
Pronuclear transfer. Supervisor: Professor Li Yunlong and Professor Chen Dayuan.
2005-2007: Postdoctoral Fellow supported by the Parkinson Society of Canada, Department
Of Cellular and Systems Biology, University of Toronto. Project: The roles of Pitx3 and
Nurr1 in specifying neural stem cells toward a dopaminergic neuronal identity. Supervisor:
Professor Vincent Tropepe.
2007-2008: Postdoctoral Fellow, Stem Cell and Cancer Research Institute, McMaster
University, Canada. Project: The isolation, proliferation and differentiation of brain
Tumor initiating cells from patient samples. Supervisor: Professor Sheila Singh.
2008-2012: Postdoctoral Fellow and Research Technician 2, Center for Research in
Neurodegenerative Diseases (CRND), Department of Medicine, University of Toronto.
Project: Lentiviral vector cloning, production, transduction and function analysis of
Parkinson disease related genes. Supervisor: Professor Anurag Tandon.
3. 2012-2014: Research Associate (Research Technician 2), Division of Nephrology,
Massachusetts General Hospital, Harvard Medical School, Boston, USA,
Project: The overexpression, and purification of Human Integrins in sf9 insect cells
for their X-Ray structures. supervisor: Professor M. Amin Arnaout.
March to April, 2015: Research Technician 2, Fred Hutchinson Cancer Research Center,
University of Washington, Seattle, USA. Project: Using CRISPR/Cas9 technology to
Generate and analyze mouse mutant cancer models; Supervisor: Professor Velari
Vasioukhin.
4. Topics
1. The “All or Half” Law of Recombinant DNA
2. Lentivirus Transduction
3. CRISPR/Cas9 for Genome Editing
4. Mouse Somatic Nuclear Transfer and Pronuclear Transfer
5. The immunogenicity and tumorigenicity of iPSCs
6. Work at Professor Yaacov Ben-David lab
5. This talk based on the following publications:
Molecular cloning:
1. Gang Zhang* & Anurag Tandon. Quantitative assessment on the
cloning efficiencies of lentiviral transfer vectors with a unique
clone site. Scientific Reports, 2012, 2: 415. IF=5.578.
2. Gang Zhang* & Anurag Tandon. Combinatorial Strategy: A highly
efficient method for cloning different vectors with various clone
sites. American Journal of Biomedical Research, 2013, 1: 112-
119
3. Editorial Comment: Gang Zhang*. A new overview on the old
topic: the theoretical analysis of “Combinatorial Strategy” for
DNA recombination. American Journal of Biomedical Research,
2013, 1(4):108-111.
4. Editorial Comment: Gang Zhang* & Yi Zhang. On the “All or
Half” Law of Recombinant DNA. American Journal of
Biomedical Research, Accepted.
6. Lentiviral transduction and protein expression:
5. Gang Zhang. Efficient lentiviral transduction of different human and mouse cells
with various genes and their mutants. Submitted to BMC Biology, IF=7.98.
iPSCs (Induced Pluoripotent Stem Cells)
6. Gang Zhang, Yi Zhang. “Mouse Clone Model” for evaluating the immunogenicity
and tumorigenicity of pluoripotent stem cells. Stem Cell Research & Therapy,
accepted, IF=3.37.
Animal cloning:
7. Gang Zhang, Qingyuan Sun, Dayuan Chen. In vitro development of mouse
somatic nuclear transfer embryos: Effects of donor cell passages and
electrofusion. Zygote, 2008, 16: 223~7, IF=1.416.
8. Gang Zhang, Qingyuan Sun, Dayuan Chen. Effects of sucrose treatment on the
development of mouse nuclear transfer embryos with morula blastomeres as
donors. Zygote, 2008, 16: 15~9, IF=1.416.
9. Kong FY*, Zhang G*, et al. Transplantation of male pronucleus derived from in
vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte
results in live offspring in mouse. Zygote, 2005, 13: 35~8 (*co-first author).
IF=1.416.
7. Topic I: The “All or Half” Law of Recombinant DNA
-Gene Cloning
8. Main topics
1. Theoretical design of combinatorial strategy
2. Special examples with BamH I clone site
3. General examples with various clone sites
9. Part I: Theoretical design of combinatorial
strategy
To clone plasmid vectors quantitatively
10. The birth of recombinant DNA technology
In 1972, Jackson et al. reported the first recombinant DNA,
SV40-λdvgal DNA was created. This work won Nobel Prize in
Chemistry in 1980 (Jackson, et al. PNAS, 1972, 69: 2904-9).
In 1973, Cohen, et al. found, for the first time, that the
recombinant DNA could be transformed into E. Coli and
biologically functional in the host. Stanford University applied
for the first US patent on recombinant DNA in 1974. This
patent was awarded in 1980 (Cohen, et al. PNAS, 1973, 70:
3240-4).
This technology revolutionarily changed the bio-medical
research during the past decades.
11. Achievements in DNA recombination:
Many different vector systems available:
1. Regular vectors: pET, pcDNA, etc.
2. Viral vectors: Adenoviral vectors, retroviral vectors, lentiviral vectors, etc.
3. Bacterium expression vectors, insect expression vectors, mammalian
expression vectors, etc.
4. Constitutive expression vectors, inducible expression vectors, etc.
5. Ubiquitous expression vectors, tissue-specific expression vectors, and so on.
At genome era, more and more gene sequences available
Therefore, in theory, we could very easily put any genes of
interest into any vectors, and transfer them into any organisms and
tissues, to investigate their functions according to our purposes.
12. Puzzles in molecular cloning:
Some times, if we are lucky, we could clone a vector easily with 5 to
10 minipreps in 3 days, in other times, if we are not lucky, we might
need to make hundreds of minipreps, and waste months for a
vector, why?
Possible reasons:
1. The sizes of the vectors and inserts;
2. The preparation methods of the inserts;
3. The ligation efficiencies of the clone sites;
4. The transformation efficiencies of the host cells, etc.
Now I want to ask “Could we find a way to clone
vectors efficiently, and quantitatively?” The answer is
YES!!!
13. ddH2O Insert Vector 10 X ligase buffer T4 DNA ligase
Add up to 20µl ~100ng ~200ng 2µl 1µl
A Mole= ~6.02 X 1023 molecules; Average Molar Weight of A, G, C, T= ~660 g
1 g=1 X 109 ng; 1 mole=1 X 1012 pmoles
Suppose the insert: 1.5kb, the vector: 5kb, then
100ng insert=100ng/(660 X 1500 X 2 X 1,000,000,000)ng=0.05pmole X 6.02 X
1023/1012 =3.01 X 1010 insert molecules
200ng vector=200ng/(660 X 5,000 X 2 X 1,000,000,000)ng=0.03pmole X 6.02 X
1023/1012=1.8 X 1010 vector molecules
So what will happen in this tiny 20µl ligation tube?
Typical reaction system of ligation
14. Main procedure of recombinant DNA
1. Choose or create compatible clone sites between
the vectors and inserts
Highly efficient clone sites, such as EcoR I, BamH I, EcoR V etc.
2. Digest and purify the vectors and inserts
The purities A260/280≥1.80
3. Ligation, high concentration T4 DNA ligase
4. Transformation, high efficient competent cells, such as DH5α,
Top10
5. Identification by restriction digestion and DNA sequencing
15. Approaches to create compatible clone sites
1. Design PCR primers contained proper clone sites for the inserts
2. Make blunt ends by Klenow fragment and T4 DNA polymerase
3. Insert clone sites by site-directed mutagenesis
16. Design PCR primers containing proper clone sites for the inserts
Advantages: easy and simple, suitable for small size regular cloning
Disadvantages: not guarantee 100% correct-cutting ends, not suitable
for large size vector cloning
From online
17. Making blunt ends for the inserts or/and vectors with Klenow
fragment or T4 DNA polymerase
Functions of Klenow fragment and T4 DNA polymerase:
1. Fill-in the 5’-overhangs to form blunt ends
2. Chew-out the 3’-overhangs to form blunt ends
3. Result in recessed ends due to the 3’ to 5’ exonuclease activity of the enzymes.
Advantages:
Easy and simple, only a short time reaction, such as 5 to 15 minutes, suitable for easy
cloning
Disadvantages:
Not guarantee 100% with the correct blunt ends, not suitable for low efficiency cloning
New England BioLabs
18. Inserting clone sites by site-directed mutagenesis (SDM)
1. Mutant strand synthesis
by PCR to
A. Denature DNA template
B. anneal mutagenic primers
containing desired mutantion
C. extend and incorporate primers with
PfuUltra DNA polymerase
2. Dpn I digestion of template
Digest methylated and
hemimethylated parental DNA with Dpn I
3. Transformation
Transform mutated molecules into
competent cells for nick repair
Stratagene SDM Kit
19. Advantages of inserting clone sites by SDM
1. The mutated products are circular double-stranded
plasmid DNA
2. The linearized inserts are theoretically 100% with
correct-cutting ends
3. Maximal ligation could achieve between the
vectors and inserts
4. Suitable for low efficiency vector cloning, such as
lentiviral vectors.
In theory, this method transformed the vector cloning
into subcloning, therefore, the efficiencies can be
improved radically.
20. The function of T4 DNA ligase
1. To catalyze the formation of 3’,
5’-Phosphodiester Bond between
juxtaposed 5’-phosphate groups
and 3’-hydroxyl groups.
2. Ligation could take place when
there are mismatches at or close to
the ligation junctions. That is to say,
T4 DNA ligase could catalyze the
ligation between different
clone sites (Haarada & Orgel, Nucl.
Acids Res., 1993, 21: 2287-91).
21. Procedure of ligation
1. Inter-molecular reaction to form
non-covalently bonded, linear
vector-insert Hybrids.
2. Intra-molecular reaction to form
non-covalently bonded, circular
molecules.
3. Annealing between the inter and
intra molecules brings the 5’-
phosphate and 3’-hydroxyl residues of
the vectors and inserts into close
alignment, which allows T4 DNA
ligase to catalyze the formation of
3’, 5’-phosphodiester bonds.
This reaction requires high
DNA concentrations
This reaction works efficiently
with low DNA concentrations.
Molecular cloning,
3rd Edition
22. 3’ 5’ 3’
Transformation and selection after DNA ligation
5’ 3
’
5’
5’
3’
Clone site A Clone site AClone site B Clone site B
Vector Insert
+
ligation
vector
Vector and insert insert
Transformation and antibiotic selection
Transformants survive Transformants survive
Transformants can
not survive
Note: clone sites A and B could be blunt ends, over-hang ends, the same or different
High efficiency Low efficiency
23. 3’-OH
The function of Calf Intestinal Phosphatase (CIP)
Vector
5’-P
5’-P3’-OH
3’-OH
CIP Treatment
3’-OH
Ligation
3’-OH
3’-OH
Can not be self-circularized
Transformation
Because the transformation efficiencies of linear DNA are very low,
the backgrounds with empty-vectors are decreased radically.
Molecular cloning, 3rd edition
24. Choosing proper competent cells for transformation
Subcloning efficiency DH5α chemical competent E. Coli:
1 X 106 CFU/µg supercoiled DNA
One shot Stbl3 chemical competent E. Coli:
1 X 108 CFU/µg supercoiled DNA
One shot Top10 chemical competent E. Coli:
1 X 109 CFU/µg supercoiled DNA
Invitrogen (Life
Technologies)
25. Theoretical design of combinatorial strategy
Enzyme digest and CIP Enzyme digest
3’-OH
3’-OH
Transformation
3’-OH
3’-OH
5’-P
5’-P
100% correct cutting, not self-ligated 100% correct cutting ends
+
Ligation
3’-OH
3’-OH
Can not circularized
Vector +
insert
insert
Top10 to increase transformation
Linerized vector
very few
Survive, 100% positive, if different clone sites
50% positive if the same and blunt clone sites
Regular or
lentiviral vector
Inserts from circular vector
with matching clone sites or
Inserted by SDM
Not survive
26. Clone sites Sizes (kb) Methods for
clone sites
Transformation
host
No. of colonies Positive
clones
Blunt sites
Small (vector<5,
insert<1.5)
Existed Top10 Dozens About 50%
large (vector>5,
insert>1.5)
Existed Top10 A few to dozens About 50%
Different
over-hang
sites
Small (vector<5,
insert<1.5)
Existed/SDM Top10 hundreds or more Nearly
100%
Large (vector>5,
insert>1.5 )
Existed/SDM Top10 Dozens to
hundreds
Nearly
100%
One over-
hang site
Small (vector<5,
insert<1.5)
SDM Top10 hundreds or more About 50%
Large (vector>5,
insert>1.5 )
SDM Top10 Dozens to
hundreds
About 50%
Predictions for molecular cloning with CIP-treated
vectors
Gang Zhang, American Journal of Biomedical Research, 2013
27. Part II: Experimental Demonstration of
combinatorial strategy with a unique BamH I
clone site for lentiviral vector cloning
28. Scheme of clone pWPI/hPlk2/Neo and pWPI/EGFP/Neo with
BamH I site
29. Identification of pWPI/EGFP/Neo digested by Not I (n=1)
Positive clones: 3, 4, 7, 9, 12, 13, 14; Negative clones: 1, 2,
5, 6, 8, 11; Clone 10 with 2 copies of insert
30. Identification of pWPI/hPlk2/Neo digested by Not I (n=1)
A: WT, 2, 4, 9, 13, 14 were positive; B: K111M, 2, 3, 6, 9,
10, 11, 12, were Positive; C: T239D, 1, 2, 7, 8, 9, 10, 12, were
positive; D: T239V, 1, 3, 5, 6, 7, 8, 9, 10, 11, 14, were positive.
31. Identification of pWPI/hPlk2 WT and mutants and pWPI/EGFP (n=3)
A, B: EGFP; B, C: hPlk2WT;
D, E: K111M; E, F: T239D;
G, H: T239V.
32. Vector
Hosts of
transformation
Total No. of
transformed
clones
Total No. of
identified
clones
Percentage of
inserted vectors
(Mean±SD)
Percentage of
Correct-oriented
inserts (Mean±SD)
EGFP Top10 149±100 (n=4) 41 (n=4) 97%±5.5%a(40) 37%±12.4% (16)
hPlk2 WT Top10 123±108 (n=4) 41 (n=4) 95%±10.5% (38) 43%±16.6% (17)
K111M Top10 123±88 (n=4) 41 (n=4) 91%±10.9% (37) 52%±21.2% (21)
T239D Top10 126±78 (n=4) 41 (n=4) 95%±6.4%a(39) 54%±9.8%a (22)
T239V Top10 98±60 (n=4) 41 (n=4) 93%±5.2% (38) 54%±12.8% (23)
Statistical analysis of Cloning efficiencies
of LVs with CIP-treated vectors (n=4)
33. Vector
Hosts of
transformation
Total No. of
identified
colonies
Percentage of
inserted vectors
Percentage of
Corrected-oriented
inserts
EGFP Top10 10 0% (0/10) 0% (0/10)
hPlk2 WT Top10 10 10% (1/10) 0% (0/10)
K111M Top10 10 10% (1/10) 0% (0/10)
T239D Top10 10 0% (0/10) 0% (0/10)
T239V Top10 10 30% (3/10) 10% (1/10)
Cloning efficiencies of LVs with non-CIP-treated
vectors (n=5)
10% 2%Total
41. Statistical analysis of cloning efficiencies of different
vectors with various clone sites
Zhang & Tandon, American Journal of Biomedical Research, 2013
42. Conclusions
Therefore, with our “Combinatorial strategy”, almost all the
plasmid vectors could be successfully cloned by “One ligation,
One transformation, and 2 to 3 minipreps”.
This is the “All or Half” law of recombinant DNA
with our method.
Clone sites Positive clones
Two different clone sites Nearly 100%
The same clone site/blunt sites About 50%
Gang Zhang & Yi Zhang, American Journal of Biomedical Research, 2015, accepted, in press.
44. Scheme of the third generation lentiviral vector system
RRE cPPT CMV GFP WPRE 3’LTR
No expression
CMV Gag-Pol pA
RSV Rev pA
CMV VSVG pA
Gag-Pol precursor protein is for integrase, reverse transcriptase and structural
proteins. Integrase and reverse transcriptase are involved in infection. Rev
interacts with a cis-acting element which enhances export of genomic transcripts.
VSVG is for envelope membrane, and lets the viral particles to transduce a broad
range of cell types. Deletion of the promoter-enhancer region in the 3’LTR (long
terminal repeats) is an important safety feature, because during reverse
transcription the proviral 5’LTR is copied from the 3’LTR, thus transferring the
deletion to the 5’LTR. The deleted 5’LTR is transcriptionally inactive, preventing
subsequent viral replication and mobilization in the transduced cells.
Tiscornia et al., Nature Protocols, 2006
pMDL
pRev
pVSVG
Transfer vector with
genes of interest
Packaging vectors
45. The advantages of the third generation lentiviral
vectors
1. LVs can transduce slowly dividing cells, and non-dividing
terminally differentiated cells;
2. Transgenes delivered by LVs are more resistant to
transcriptional silencing;
3. Suitable for various ubiquitous or tissue-specific
promoters;
4. Appropriate safety by self-inactivation;
5. Transgene expression in the targeted cells is driven solely
by internal promoters;
6. Usable viral titers for many lentiviral systems.
Naldini et al., Science, 1996; Cui et al., Blood, 2002; Lois et al, Science, 2002
46. The disadvantages of the third generation
lentiviral vectors
1. Lentiviral vectors are self-inactivated by the deleting
of 3’-LTR region, therefore, they can not be replicated
in host cells. For each lentiviral vector, the titer is
solely dependent on the transfection step;
2. Only the host cells co-transfected with all the four
vectors, can produce lentiviral particles for infection;
3. To make efficient lentiviral transduction, good tissue
culture and transfection techniques are very
important, such as lipofectamine transfection.
47. Lentiviral vector system in our research
Transfer vectors:
pWPI-Neo
pLenti-CMV/TO-Puro-DEST
Packaging vectors:
EF1-α Gene IRES Neomycin
attR1-CmR-ccdB-attR2
PuromycinCMV/TO Gene PGK
About 11.4kb
About 1.7 kb, this is for GateWay cloning, we
cut off this sequence in our cloning
About 7.8kb
VSV-GCMV
Tat/RevGag/PolCMV pPAX2
pMD2.G
In order to get sufficient titers, we used the third generation of transfer vectors
and the second generation of packaging vectors to produce lentiviruses.
Campeau et al., PLoS One, 2009
48. Working procedure of lentiviral transduction:
293T cells in DMEM + 10% FBS
Co-transfected with lentiviral transfer
vectors, pM2D and pPAX2 packaging
vectors by Lipofectamine
Transfection
Collect supernatants
with lentiviruses, 48-72 hours later
Infection of
targeting cells
293, SHSY5Y, NSC, BV-2, etc.
Selection
2 weeks
Stable cell lines with transgenes
49. The scheme of titration using 6-well plates
10-2
900μl
293 cells
10-3
900μl
293 cells
10-4
900μl
293 cells
10-5
900μl
293 cells
10-6
900μl
293 cells
10-7
1000μl
293 cells
100μl 100μl 100μl
100μl 100μl 100μl
Titers (TU/ml)=colony number X dilution factors X (1000/900) (if not
the last well) to transform into 1 ml volume.
1. Let 293 cells are about 70-80% confluence when infect.
2. Using suitable drugs to select for 2 weeks, such as G418, Puromycin, etc.
3. Dye the live cell colonies with crystal violet, and count the blue colonies.
50. The advantages of our titration method
1. It is convenient to calculate the titers;
2. Using large volume for 10 fold dilution can decrease
the deviation;
3. Compared with other titration methods, it is more
informative for next-step infection.
51. pLenti-CMV/TO-β5 WT-Puro titration (n=3)
10-210-4
10-3
10-7 10-6
10-5
A B
C
A: 16 X 105 X10/9=1.78 X 106 TU/ml
B: 9 X 105 X 10/9=1.0 X 106 TU/ml
C: 19 X 105 X 10/9=2.1 X 106 TU/ml
Mean ± SD=1.63 X 106 ± 5.66 X 105 TU/ml
10-4
10-4
10-5
10-5
Choose the suitable wells for counting
52. pLenti-CMV/TO-β5 T1A-Puro titration (n=3)
A: 44 X103 X 10/9=4.89 X 104 TU/ml
B: 67 X103 X 10/9=7.44 X 104 TU/ml
C: 25 X103 X 10/9=2.78 X 104 TU/ml
Mean ± SD=5.04 X 104 ± 2.33 X 104
TU/ml
Note: 100 X lower than WT
A B
C
10-310-2
10-4
53. Titration of pWPI/Neo, pWPI-EGFP-Neo, and pWPI-
hPlk2WT/Neo
A B
C
10-6
10-4
10-4
A: pWPI-Neo
B: pWPI-EGFP-Neo
C: pWPI-hPlk2WT-Neo
54. A B
C
Titration of pWPI-hPlk2K111M-Neo, pWPI-hPlk2T239D-
Neo, and pWPI-hPlk2T239V-Neo
A: pWPI-hPlk2K111M-Neo;
B: pWPI-hPlk2T239D-Neo;
C: pWPI-hPlk2T239V-Neo.
10-4
10-4
10-4
58. A B
C A: Lipo-transfection of 293
cells with CMV-DsRed plasmid;
B: Lipo-transfection of 293
cells with EF1α-EGFP plasmid;
C: Mouse neural stem cells
from the midbrain of D14.5
fetus.
59. A B
C A: Adult mouse whole brain neural
progenitor cells (NPCs) infected with
pWPI-Neo lentiviral vector. B: NPCs
infected with pWPI-β5WT-IRES-Neo
lentiviral vector. C: NPCs infected with
pWPI-β5T1A-IRES-Neo lentiviral vector.
All are selected with G418, at P12.
60. The mechanism of tetracycline-regulated
inducible expression system
PCMV TetR BlasticidinpLenti/TR
TetR protein
1. Express Tet Repressor
protein in mammalian
cells, TetON cell lines
2. To form TetR
homodimers
PuromycinGENETetOTetOPCMV
X
Expression repressed
3. TetR dimers bind with TetO
sequence to repress the expression
pLenti/CMV/TO
61. PCMV TetO TetO GENE Puromycin
4. added Tet binds to
TetR homodimers
PCMV TetO TetO GENE Puromycin
5. The binding of Tet and TetR
dimers causes a conformational
change in TetR, release from the Tet
operator sequences, and induce the
expression of gene of interest.
Expression derepressed
Modified from Invitrogen
62. TetraHis GAPDH
1 2 3 4 5 6 7 8 9 10
Western blot for SHSY5YTO/pLentiTO/β5WT induced
expression with 1µg/ml DOX for different days (12%, 1mm gel)
Lane 1: Marker; Lane 2: not induced; Lane 3: induced Day 1;
Lane 4: Day 2; Lane 5: Day 3; Lane 6: Day 4;
Lane 7: Day 5; Lane 8: Day 6; Lane 9: Day 7;
Lane 10: Day 8.
63. TetraHis GAPDH
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
Western blot for SHSY5YTO/pLentiTO/β5WT
induced expression with different concentrations
of DOX for 5 days (12%, 1mm gel)
Lane 1: Marker; Lane 2: not induced; lane 3: 0.005µg/ml;
Lane 4: 0.01 µg/ml; Lane 5: 0.05µg/ml; Lane 6: 0.1µg/ml;
Lane 7: 1µg/ml; Lane 8: SHSY5YTO cells for negative control;
Lane 9: SHSY5YTO/pLentiTO/β5WT, Positive control.
64. Western blot for SHSY5YTO/pLenti-β5T1A induced
expression with 1µg/ml Dox for different days (12% Gel,
1.00mm)
Lane M: Marker;
Lane 1: T1A not induced; Lane 2: T1A 1µg/ml Dox, Day 1;
Lane 3: Day 2; Lane 4: Day 3; Lane 5: Day 4; Lane 6: Day 5;
Lane 7: Day 6; Lane 8: Day 7; Lane 9: Day 8.
M 1 2 3 4 5 6 7 8 9 M 1 2 3 4 5 6 7 8 9
Tetra-His GAPDH
Gang Zhang, 2015, Submitted
65. TetraHis GAPDH
M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8
Western blot for SHSY5YTO/pLentiTO/β5T1A
induced expression at different concentration of
DOX for 5 days (12%, 1.00mm gel)
Lane M: Marker;
Lane 1: T1A Mock (not induced); Lane 2: 0.005µg/ml DOX;
Lane 3: 0.01µg/ml; Lane 4: 0.05µg/ml; Lane 5: 0.1µg/ml;
Lane 6: 1µg/ml;
Lane 7: SHSY5YTO cells, Negative control;
Lane 8: SHSY5YTO/pLentiTO/β5T1A Positive control
66. 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Myc GAPDH
Ubiquitin
The relationship between the induced (50ng/ml DOX)
expression of β5WT and β5T1A with cell ubiquitin levels
(7.5% gel) (n=2)
Lane 1: marker; Lane 2: β5WT D0; lane 3: β5WT D7;
lane 4: β5T1A D0; lane 5: βT1A D7; Lane 6: β5WT D0’;
lane 7: β5WT D7’; lane 8: β5T1A D0’; lane 9: β5T1A D7’;
Lane 10: SHSY5YTO cells, negative control.
67. Myc GAPDH Ubiquitin
1 2 3 4 5 6
The relationship between the induced (50ng/ml DOX)
expression of β5WT and β5T1A with cell ubiquitin levels
(7.5% gel) (n=1)
Lane 1: marker; Lane 2: β5WT D0’’; lane 3: β5WT D7’’;
lane 4: β5T1A D0’’; lane 5: β5T1A D7’’;
Lane 6: SHSY5YTO cells, negative control
68. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
WT D0 WT D7 T1A D0 T1A D7 Nega
Myc/GAPDH
0
10
20
30
40
50
60
70
WT D0 WT D7 T1A D0 T1A D7 Nega
Ubiquitin/GAPDH
The induced expression of
β5WT and β5T1A (n=3).
The relationship between
induced expression of
β5WT and β5T1A with the
level of Ubiquitin
expression (n=3).
SHSY5YTO cells are at
“growing state”.
69. 1 2 3 4 5 6 7 8 9 10
A B C
TetraHis
α-Syn
GAPDH
The induced expression of β5WT (1µg/ml DOX) in SHSY5YTetOn
cells and the endogenous expression of α-Syn.
Lane 1: Marker; lane 2: D0; lane 3: D1; lane 4: D2; lane 5: D3; lane 6: D4; lane 7:
D5; lane 8: D6; lane 9: D7; lane 10: SHSY5YTetOn cells.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Negative
TetraHis/G
APDH
Syn-
1/GAPDH
70. 1 2 3 4 5 6 7 8 9 10
Syn-1 Myc
GAPDH
The relationship between induced expression of β5WT and β5T1A
with the endogenous expression level of α-Synuclin (15% Gel)
(n=2).
Lane 1: marker; Lane 2: β5WT D0; lane 3: β5WT D7; lane 4: β5T1A D0;
lane 5: β5T1A D7; EXP#1. Lane 6: β5WT D0’; lane 7: β5WT D7’; lane 8: β5T1A D0’;
lane 9: β5T1A D7’; Exp#2. Lane 10: SHSY5YTO cells, negative control.
71. 1 2 3 4 5 6
Syn-1 Myc GAPDH
The relationship between induced expression of β5WT
and β5T1A with the endogenous expression level of α-
Synuclin (15% Gel) (n=1).
Lane 1: marker; Lane 2: β5WT D0’’; lane 3: β5WT D7’’;
lane 4: β5T1A D0’’; lane 5: β5T1A D7’’; EXP#3.
Lane 6: SHSY5YTO cells, negative control.
72. A
11A5
(phosphorylated α-Syn)
B
Myc
(β5WT, β5T1A)
C
GAPDH
1 2 3 4 5 6
Expression of endogenous phosphorylated α-Syn and
induced β5WT and β5T1A
Lane 1: Marker; lane 2: β5WT D0; lane 3: β5WT D7;
lane 4: β5T1A D0; lane 5: β5T1A D7;
Lane 6: negative control.
73. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
WT D0 WT D7 T1A D0 T1A D7 Negative
Myc/GAPDH
0
0.005
0.01
0.015
0.02
0.025
WT D0 WT D7 T1A D0 T1A D7 Negative
11A5/GAPDH
Statistical analysis of the relationship between induced
expression of β5WT and β5T1A with the endogonous
expression of phosphorylated α-Syn (n=3)
74. My lentiviral transductin and expression work
contributed to the following papers:
1. N. Visanji, S. Wislet-Gendebien, L. Oschipok, G. Zhang, I. Aubert, P. Fraser, A.
Tandon. The effect of S129 phosphorylation on the interaction of alpha-
synuclein with synaptic and cellular membranes. The Journal of Biological
Chemistry, 2011, 286: 35863-35873.
2. Robert HC Chen, Sabine Wislet-Gendebien, Filsy Samuel, Naomi P Visanji,
Gang Zhang, Marsilio D, Tanmmy Langman, Paul E Fraser, and Anurag
Tandon. Alpha-synuclein membrane association is regulated by the Rab3a
recycling machinery and presynaptic activity. The Journal of Biological
Chemistry, 2013, (Selected as the Journal of Biological Chemistry "Paper of
the Week“.
75. Topic III: CRISPR/Cas9 Technology for Genome Editing
CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeat)/Cas9 (CRISPR-associated)
sgRNA (single guide RNA)
Plasmid DNA with targeting sequences
2 pronucleus
1-cell fertilized mouse egg
Microinjection of Cas9 RNA,
sgRNA, and plasmid DNA
together, can edit the genome
at specific genome locations
Yang et al., Cell, 2013, 154: 1370-9
76. Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M.
Generation of mutant mice by pronuclear injection of circular
plasmid expressing Cas9 and single guided RNA. Sci Rep. 2013,
3:3355.
1. Making Cas9 and sgRNAs by in vitro transcription;
2. Transfecting different plasmid combinations into 293FT cells to
confirm negative and positive controls for in vivo mouse genome
editing models
This is part of my work in Fred Hutchinson Cancer Research Center,
University of Washington
77. Topic 4: Mouse Nuclear Transfer and Pronuclear Transfer
1. Gang Zhang, Qingyuan Sun, Dayuan Chen. In vitro development
Of mouse somatic nuclear transfer embryos: Effects of donor cell
Passages and electrofusion. Zygote, 2008, 16: 223-7.
2. Gang Zhang, Qingyuan Sun, Dayuan Chen. Effects of sucrose
treatment on the development of mouse nuclear transfer embryos
with morula blastomeres as donors. Zygote, 2008, 16: 15-9.
3. Kong FY*, Zhang G*, et al. Transplantation of male pronucleus
derived from in vitro fertilization of enucleated oocyte into
parthenogenetically activated oocyte results in live offspring in mouse.
Zygote, 2005, 13: 35-8 (* Co-first author).
This is my PH. D work in Institute of Zoology, Chinese Academy of
Sciences
78. Procedure of NT manipulation with mouse ear fibroblasts as donors
81. The in vitro fertilization of Kunming (white) female
mouse enucleated MⅡoocytes with the capacitated
sperms of C57BL/6 male mouse
82. Manipulation process of mouse
pronuclear transplantation
A and B:
Cutting the zona pellucida of Kunming
(white) mouse parthenogenetically
activated embryo
C and D:
Aspirating the male pronucleus
of C57BL/6 mouse in vitro fertilized
Embryo;
E and F:
Transferring the male
Pronucleus of C57BL/6 mouse into
the perivitelline space of Kunming
(white) mouse parthenogenetically
activated embryo.
83. Confocal microscopy (PI staining)
A: In in vitro fertilized embryo, two male pronuclei were observed obviously
with red color;
B: In parthenogenetically activated embryo, a single female pronucleus was
observed obviously with red color.
84. Embryo transfer and fetuses born of mouse pronuclear transplantation embryos
No. of
experiment
No. of embryos
transferred
No. of
recipients
Transfer
sites
No. of
pregnancy
No. of fetuses
born
22 381 22 Oviducts 1 7
Offsprings of pronuclear transplantation and their
foster mother
85. Immunogenicity and tumorigenicity of iPSCs
1. In 2006, Takahashi & Yamanaka demonstrated that induced pluripotent
stem cells (iPSCs) can be generated from mouse embryonic or adult
fibroblasts by overexpression four factors, Oct3/4, Sox2, c-Myc, and
Klf4, under ES cell culture conditions (Cell, 2006, 126: 663-676). This
discovery won Nobel Prize in 2012 in Physiology and Medicine.
2. In 2009, Gao & Zhou groups proved that iPSCs can suport the full
development of mice to term by tetraploid complementation (Kang, et
al., Cell Stem Cell, 2009, 5:135-138; Zhao et al., Nature, 2009, 461:86-
90). Therefore, demonstrated that iPSCs have the full developmental
potentials the same as ESCs.
3. Hence, iPSCs are the most promising cells for stem cell therapy,
because they overcome the barrier of the ethical concerns from using
the embryos, can provide sufficient sources for transplantation and give
a bright hope for stem cell therapy by isolating patient-derived iPSCs.
87. The limitations for iPSCs transplantation therapy
However, due to the possibility that iPSCs can give rise to
cancers, and induce immune rejection reactions based on the
previous and current investigations, iPSCs will be useless,
clinically, in stem cell therapy, unless we can create a new
suitable model to prove that, the iPSCs can not form cancer and
induce immune rejection when they are transplanted into the
same donors.
Based on this need, we designed a new mouse Model, called
“Mouse Clone Model” to address this problem.
88. “Mouse Clone Model” for evaluating the
immunogenicity and tumorigenicity of iPSCs
Gang Zhang & Yi Zhang, Stem Cell Research & Therapy, Accepted.
89. Acknowledgement
The Parkinson Society of Canada grant (The Margaret Galloway
Basic Research Fellowship) to Gang Zhang. (2005-2007)
The Stem Cell Network of Canada grant to Dr. Vincent Tropepe
(2005-2007), Department of Cellular & Systems Biology, U of T
The Canadian Institutes of Health Research (CIHR) grant
MOP84501 and the Parkinson Society of Canada grant to
Dr. Anurag Tandon, Centre in Research for Neurodegenerative
Diseases (CRND), U of T.
Thank you all for the attendance!
