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!