On the All or Half Law of Recombinant DNA, Lentivus Transduction and some others.pptx - Copy
-
Upload
gang-zhang -
Category
Documents
-
view
220 -
download
0
Transcript of On the All or Half Law of Recombinant DNA, Lentivus Transduction and some others.pptx - Copy
On the “All or Half” law of Recombinant DNA, Lentivirus Transduction and Some Others
Gang Zhang, Ph. D
Research Technician IICentre for Research in Neurodegenerative DiseasesDepartment of MedicineUniversity of Toronto, Canada
Main Research Experience2001-2005: 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, DepartmentOf 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 brainTumor 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.
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 cellsfor 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 toGenerate and analyze mouse mutant cancer models; Supervisor: Professor Velari Vasioukhin.
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
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.
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.
Topic I: The “All or Half” Law of Recombinant DNA-Gene Cloning
Main topics
1. Theoretical design of combinatorial strategy
2. Special examples with BamH I clone site
3. General examples with various clone sites
Part I: Theoretical design of combinatorial strategy
To clone plasmid vectors quantitatively
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.
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 ofinterest into any vectors, and transfer them into any organisms andtissues, to investigate their functions according to our purposes.
Puzzles in molecular cloning:
Some times, if we are lucky, we could clone a vector easily with 5 to10 minipreps in 3 days, in other times, if we are not lucky, we mightneed 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!!!
ddH2O Insert Vector 10 X ligase buffer T4 DNA ligaseAdd 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
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
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
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
Making blunt ends for the inserts or/and vectors with Klenowfragment 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 easycloning
Disadvantages:
Not guarantee 100% with the correct blunt ends, not suitable for low efficiency cloning
New England BioLabs
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
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.
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 differentclone sites (Haarada & Orgel, Nucl. Acids Res., 1993, 21: 2287-91).
Procedure of ligation
1. Inter-molecular reaction to formnon-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
3’ 5’ 3’
Transformation and selection after DNA ligation5’ 3’ 5’
5’ 3’
Clone site A Clone site AClone site B Clone site B
Vector Insert+
ligation
vectorVector and insert insert
Transformation and antibiotic selection
Transformants survive Transformants surviveTransformants can not survive
Note: clone sites A and B could be blunt ends, over-hang ends, the same or different
High efficiency Low efficiency
3’-OH
The function of Calf Intestinal Phosphatase (CIP)Vector
5’-P5’-P3’-OH
3’-OH
CIP Treatment
3’-OH
Ligation
3’-OH3’-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
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)
Theoretical design of combinatorial strategy
Enzyme digest and CIP Enzyme digest3’-OH
3’-OH
Transformation
3’-OH3’-OH
5’-P5’-P
100% correct cutting, not self-ligated 100% correct cutting ends
+
Ligation
3’-OH3’-OH
Can not circularizedVector + insert
insert
Top10 to increase transformation
Linerized vector very few
Survive, 100% positive, if different clone sites50% positive if the same and blunt clone sites
Regular orlentiviral vector
Inserts from circular vectorwith matching clone sites orInserted by SDM
Not survive
Clone sites Sizes (kb) Methods for clone sites
Transformation host
No. of colonies Positive clones
Blunt sitesSmall (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
Part II: Experimental Demonstration of combinatorial strategy with a unique BamH I clone site for lentiviral vector cloning
Scheme of clone pWPI/hPlk2/Neo and pWPI/EGFP/Neo with BamH I site
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
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.
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.
VectorHosts 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)
VectorHosts 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
Inserted clones Positive clones0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
CIPedUn-CIPed
Cloning efficiencies of LVs with CIP-treated and un-CIP-treated vectors with BamH I site
Zhang & Tandon, Scientific Reports, 2012, 2: 415
Transient expression of hPlk2 Wt and mutants and EGFPin 293T cells
1, 6: 293T cells;2, 3, 4, 5: hPlk2Wt;7, 8: K111M;9, 10: T239D;11, 12: T239V;c, e: EGFP
Vector sizes: ~13kb
Part III: General examples for different vector cloning with various clone sites
Scheme of cloning LVs with blunt clone sites (Swa I, EcoR V, Pme I)
Scheme of cloning pLVCT LVs with one blunt site and another overhang Pst I site
Scheme of cloning different vectors with two different overhang sites and one Xba I site
Two different clone sites
One unique clone site
Vector & clone sites Inserts & clone sites Transformed colonies
Inserted colonies
Positive colonies
pWPI (Swa I) α-Syn-WT (Pme I) 13 (n=1) 3 (75%) 1 (25%)pWPI (Swa I) α-Syn-A30P (Pme I) 7 (n=1) 4 (100%) 3 (75%)pWPI (Swa I) α-Syn-A53T (Pme I) 10 (n=1) 1 (25%) 1 (25%)pWPI (Swa I) Rab-WT (Pme I) 2 (n=1) 2 (100%) 2 (100%)pWPI (Swa I) Rab-T36N (Pme I) 14 (n=1) 8 (80%) 2 (20%)pWPI (Swa I) Rab-Q (Pme I) 11 (n=1) 4 (100%) 3 (75%)pWPI (Swa I) GDI-WT (Pme I) 13 (n=1) 4 (66.7%) 3 (50%)pWPI (Swa I) GDI-R218E (Pme I) 7 (n=1) 2 (40%) 1 (20%)pWPI (Swa I) GDI-R (Pme I) 10 (n=1) 4 (100%) 1 (25%)pWPI (Swa I) β5-WT (EcoR V, Pme I) 20 (n=1) 2 (100%) 2 (100%)pWPI (Swa I) β5-T (EcoR V, Pme I) 2 (n=1) 1 (50%) 1 (50%)pLenti (EcoR V) β5-WT (EcoR V, Pme I) 12 (n=1) 6 (75%) 3 (37.5%)pLenti (EcoR V) β5-T (EcoR V, Pme I) 13 (n=1) 7 (87.5%) 1 (12.5%)pLVCT (Pme I, Pst I) β5-WT (EcoR V, Pst I) ~300 (n=1) 4 (80%) 4 (80%)pLVCT (Pme I, Pst I) β5-T (EcoR V, Pst I) ~100 (n=1) 5 (100%) 5 (100%)pcDNA4 (Not I, Xho I) β5-WT (Not I, Xho I) ~500 (n=1) 8 (100%) 8 (100%)pTet (Xba I) β5-WT (Xba I) ~1000 (n=1) 14 (100%) 4 (28.6%)pTet (Xba I) β5-T1A (Xba I) ~1000 (n=1) 14 (100%) 6 (42.9%)
Cloning efficiencies of different vectors with variousclone sites
Statistical analysis of cloning efficiencies of different vectors with various clone sites
Zhang & Tandon, American Journal of Biomedical Research, 2013
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 clonesTwo 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.
Topic II: Lentiviral Production, titration, and expression in mammalian cells
Scheme of the third generation lentiviral vector system
5’LTR 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 withgenes of interest
Packaging vectors
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
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.
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 vectorsand the second generation of packaging vectors to produce lentiviruses.
Campeau et al., PLoS One, 2009
Working procedure of lentiviral transduction:293T cells in DMEM + 10% FBSCo-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.Selection2 weeks
Stable cell lines with transgenes
The scheme of titration using 6-well plates
10-2
900μl293 cells
10-3
900μl293 cells
10-4
900μl293 cells
10-5
900μl293 cells
10-6
900μl293 cells
10-7
1000μl293 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.
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.
pLenti-CMV/TO-β5 WT-Puro titration (n=3)
10-2 10-4 10-3
10-7 10-6 10-5
A B
CA: 16 X 105 X10/9=1.78 X 106 TU/mlB: 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
pLenti-CMV/TO-β5 T1A-Puro titration (n=3)
A: 44 X103 X 10/9=4.89 X 104 TU/mlB: 67 X103 X 10/9=7.44 X 104 TU/mlC: 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
10-3 10-2 10-4
Titration of pWPI/Neo, pWPI-EGFP-Neo, and pWPI-hPlk2WT/Neo
A B
C
10-6
10-4
10-4A: pWPI-NeoB: pWPI-EGFP-NeoC: pWPI-hPlk2WT-Neo
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.
10-410-4
10-4
pWPI pWPI/α-SynWT pWPI/α-SynA30P pWPI/α-SynA53T0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
50000000
Lenti
vira
l Tite
rs
4.18 X 107
± 9.58 X 106
2.55 X 107
± 5.83 X 106
4.55 X 107
± 3.12 X 107
4.58 X 107
± 9.04 X 106
Statistical analyses of the Titers of pWPI empty vector, pWPI/α-SynWT, pWPI/α-SynA30P, and pWPI/α-SynA53T (n=3)
pWPI/EGFP pWPI/hPlk2WT pWPI/hPlk2K111M
pWPI/hPlk2T239D
pWPI/hPlk2T239V
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
Lenti
vira
l Tite
rs
3.33 X 106
± 3.85 X 106 4.96 X 106
± 1.33 X 106
6.92 X 106
± 2.87 X 106
3.55 X 106
± 1.98 X 106 3.04 X 106
± 2.07 X 106
Statistical analyses of Titers of pWPI/EGFP, pWPI/hPlk2WT, pWPI/K111M, pWPI/2T239D, pWPI/T239V (n=3)
pWPI/β5WT pWPI/β5T1A pWPI/GDIR218E
pWPI/GDIR240A
pWPI/Rab3AWT
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
Lenti
vira
l Tite
rs
3.11 X 107
± 1.49 X 107
1.6 X 107
± 5.14 X 106
3.26 X 107
± 8.22 X 106
2.44 X 107
± 2.25 X 106
3.93 X 107
± 3.86 X 106
Statistical analyses of Titres of pWPI/β5WT, pWPI/β5T1A, pWPI/GDIR218E, pWPI/GDIR240A, and pWPI/Rab3AWT (n=3)
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.
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.
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
XExpression repressed
3. TetR dimers bind with TetO sequence to repress the expression
pLenti/CMV/TO
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
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.
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.
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
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
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.
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
WT D0 WT D7 T1A D0 T1A D7 Nega0
0.05
0.1
0.15
0.2
0.25M
yc/G
APDH
WT D0 WT D7 T1A D0 T1A D7 Nega0
5
10
15
20
25
30
35
40
45
50
Ubiq
uitin
/GAP
DHThe 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”.
1 2 3 4 5 6 7 8 9 10A B C
TetraHisα-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.
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Negative0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
TetraHis/GAPDHSyn-1/GAPDH
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.
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.
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.
WT D0 WT D7 T1A D0 T1A D7 Negative0
0.2
0.4
0.6
0.8
1
1.2
1.4
Myc
/GAP
DH
WT D0 WT D7 T1A D0 T1A D7 Negative0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
11A5
/GAP
DH
Statistical analysis of the relationship between induced expression of β5WT and β5T1A with the endogonous expression of phosphorylated α-Syn (n=3)
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“.
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 DNAtogether, can edit the genomeat specific genome locations
Yang et al., Cell, 2013, 154: 1370-9
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
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 cellPassages 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 embryoswith 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
Procedure of NT manipulation with mouse ear fibroblasts as donors
Procedure of NT with morula blastomeres as donors
Manipulation process of mouse MⅡoocyte enucleation
The in vitro fertilization of Kunming (white) female mouse enucleated M oocytes with the capacitated Ⅱ
sperms of C57BL/6 male mouse
Manipulation process of mouse pronuclear transplantation
A and B: Cutting the zona pellucida of Kunming(white) mouse parthenogeneticallyactivated embryo
C and D: Aspirating the male pronucleusof 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.
Confocal microscopy (PI staining)A: In in vitro fertilized embryo, two male pronuclei were observed obviouslywith red color; B: In parthenogenetically activated embryo, a single female pronucleus was observed obviously with red color.
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
Immunogenicity and tumorigenicity of iPSCs1. 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.
Applications of iPS Cell Technology
Shinya Yamanaka, Cell, 2009
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.
“Mouse Clone Model” for evaluating the immunogenicity and tumorigenicity of iPSCs
Gang Zhang & Yi Zhang, Stem Cell Research & Therapy, Accepted.
Acknowledgement
The Parkinson Society of Canada grant (The Margaret GallowayBasic 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!