1. Ockunzzi 1
OCKUNZZI, Jeremiah David
Thyroxine Binding Globulin (TBG) as a Liver-Specific Promoter for Inducing SDF-1 Expression
in Trans-gene Therapy.

2. Ockunzzi 2
1. Abstract
Stromal cell-derived factor 1 (SDF-1), a chemokine has been shown to mediate stem cell
homing. Cells overexpressing SDF-1 activate the homing of stem cells, which differentiate into
the cells found in the target tissue. In these cell lines, induced stem cell homing stimulate tissue
regeneration and repair in, tested in ischemic cardiac myocytes post-infarct. The promoter for
thyroxine binding globulin (TBG) was tested as a liver specific promoter to induce SDF-1
expression in liver (HepG2) cells, in an attempt to replicate tissue repair shown in the heart. The
TBG promoter was cloned into bacterial plasmids and tested against other promoter-enhancer
combinations in three cell lines (heart- H9c2, kidney- 293A, and liver- HepG2) to test its
strength, specificity, and ability to drive SDF-1 expression. TBG was shown to be a weak
promoter in comparison to the constitutive promoter for cytomegalovirus (CMV), and its
expression was increased when coupled with the Ru5 translational enhancer element. TBG
showed expression exclusively in liver (HepG2) cells, and successfully drove SDF-1 expression
in the HepG2 cell line. SDF-1 expression in the liver has induced stem cell homing and
differentiation into albumin producing cells. Coupled with the TBG promoter, vectors containing
the SDF-1 protein encoding region transfected into liver cells, should activate stem cell homing
to the liver and cause tissue repair, further tests would test this conjecture.

3. Ockunzzi 3
2. Introduction
The latest surveys conducted by the American Liver Foundation found more than 30
million Americans living with major liver disease, making liver-targeted regenerative therapy an
attractive field for research. Damaged or diseased livers cause immune deficiencies, metabolic
complications, and reduced effectiveness in blood purification (American Liver Foundation
2015), potentially life threatening conditions if not treated.
A major focus in regenerative treatments has become the use of stem cells in therapeutic
medicine, with much attention given to the utilization of a patient’s own bone marrow-derived
stem cells for treatment (Rafi and Lyden 2003, Prockop 1997, Amado et al. 2005). Among the
many stem cell-related therapies implemented, the use of stromal cell-derived factor-1 (SDF-1)
has yielded promising results. Injured brain and heart tissues have been found to express SDF-1
after cell damage (Askari et al. 2003), leading to the recruitment of progenitor stem cells which
begin to process of tissue regeneration and healing (Lau and Wang 2011).
Overexpression of SDF-1 in cardiomyocytes, the cells where the effects of SDF-1 have
been the most extensively studied, has been found to enhance the natural tissue repair processes
induced by its presence. SDF-1 recruited stem cells also prevent cell death, improve cardiac
myocyte survival, and remodel ventricular tissue in ischemia-damaged cardiac tissues
(Sundararaman et al. 2011). Acting on CXCR4 receptors, SDF-1 causes the homing of bone
marrow-derived stem cells, which then differentiate into the targeted tissue cells (Penn et al.
2012). The CXCL12 gene, which encodes the SDF-1 protein, is expressed in the majority of
tissues throughout the body, including hepatocytes, whose expression of CXCL12 is even greater
than in cardiac cells (Gene Report-CXCL12 gene 2016). While the effects of SDF-1 in liver

4. Ockunzzi 4
cells has not been studied as extensively as in heart cells, local injection of the SDF-1 protein
into mouse livers increased CXCR4 activation and mediated homing of stem cells which
subsequently differentiated into cells producing albumin, a major secretion of hepatocytes
(Kollet et al. 2003). The activation of CXCR4 receptors by SDF-1 overexpression in the liver
suggests that with the use of a tissue-specific promoter, the regenerative therapeutics carried out
in cardiac tissue can be replicated in hepatic cells.
The use of a tissue-specific promoter restricts expression of the transgene delivered to the
targeted cells. For this reason it is necessary to use a gene encoding for a local protein (Zheng
and Baum 2009). For liver-specific gene therapies, the promoter for the thyroxine binding
globulin (TBG) gene has proven effective as a promoter for delivering transgenic expression to
hepatic cells (Yan et al. 2012). TBG synthesis occurs in the liver, making the use of its promoter
sequence viable for transgene therapy directed toward hepatic regeneration. SDF-1 genes
delivered to liver cells in vectors containing the TBG promoter should cause hepatic cells to
overproduce the SDF-1 protein. Overproduction of SDF-1 causes increased activation of CXCR4
receptors (Penn et al. 2012, Pilarisetti and Gupta 2001, Askari et al. 2003), which should then
cause stem cell homing to the liver (Kollet et al. 2003). The purpose of this study was to test how
well the TBG promoter drove expression of trans-gene vectors, and whether or not it could
induce SDF-1 production in hepatocytes, setting up future experiments to test if this SDF-1
production could cause stem-cell homing and how well the activated progenitor cells act in
regenerative therapies in the liver.
3. Materials and Methods
3.1 Plasmid Construction

5. Ockunzzi 5
Human genomic DNA gDNA was isolated and PCR was used to amplify the hTBG
promoter (GenBank ID:L13470, region -435 to -26 from TSS) utilizing the following primers
with added BglII and SpeI restriction sites. The PCR products were run on a 1% agarose gel at
100V, 100mA for 1hr (Fig. 1A).
TBG Forward Primer: 5’-GCCGATACGAGATCTCCTAGGGAGTCCTGCATGTATAATTTCTACAGAACCTATTAGA-3’
TBG Reverse Primer:5’-GGTACCGGCACTAGTGGTCACCGATGGCAAGGACGGTGATTTATAGCATGTCCTGTATTGCAAACTAGA-3’
The hTBG product was then ligated into a pCR2.1 vector (Invitrogen, Carlsbad, CA,
USA). Treatment vectors containing altering combinations of the CMV promoter, Ru5 enhancer
elements, and the Luciferase gene, which were obtained from the KCP Luc vector (Penn Labs,
Rootstown, OH, USA). These treatment vectors were constructed through the use of restriction
enzyme digests- The hTBG pCR2.1 vector and the KCP Luc vector were each sequentially
digested with BglII and SpeI-HF restriction enzymes, releasing the hTBG Promoter and CMV
promoter region, respectively. The hTBG Promoter was then ligated into the KCP Luc vector,
the post-ligation DNA was run on a 1% agarose gel to confirm the presence of the TBG
promoter in the vector (Fig. 1B).
The hTBG Ru5 Luc vector was then digested with SpeI-HF and HindIII-HF to remove its
enhancer region and was then blunted and ligated back on itself, the post-ligation DNA was run
on a 1% agarose gel (Fig. 1C). The KCP Luc vector was used to construct the CMV Luc, Ru5
Luc, and Luc vectors by using restriction sites to remove elements before ligating the vector
closed again. The KCP Luc vector itself was used as a treatment vector as well.
Construction of the vectors containing stromal-cell-derived factor 1 (SDF-1) was
accomplished through digesting a JVS-100 vector (property of Juventas Therapeutics, and patent
protected) sequentially with BglII and SpeI-HF, releasing the promoter region of the plasmid.
The hTBG PCR product was then ligated into the vector. This vector was then taken and

6. Ockunzzi 6
simultaneously digested with SpeI-HF and HindIII-HF to remove its enhancer region and then
blunted and ligated back closed (Fig. 2A).
3.2 Cell culture
The Human hepatoblastoma-derived (HepG2) cells were cultured in Eagle’s Minimum
Essential Medium (EMEM), supplemented with 10% fetal bovine serum (FBS), 1% 100x
penicillin streptomycin, and maintained in a humidified 37°C incubator containing 5.0% CO2
(Bokhari et al. 2007). Heart-derived myogenic cells (H9C2) were cultured in Dulbecco’s
Modified Eagle Medium (DMEM) with 10% FBS and 1% 100x penicillin streptomycin added to
make serum media. H9C2 cells were kept in a humidified 37°C incubator containing 5.0% CO2
(Ekhterae et al. 1999). Human embryonic kidney 293 (293A) cells were cultured in DMEM,
supplemented with 10% FBS, 1% 100x penicillin streptomycin, and 0.1mM MEM non-essential
amino acids (NEAA) added to make serum media. 293A cells were kept in a humidified 37°C
incubator containing 5.0% CO2 (Li et al. 2015).
3.3 Transfection and Dual Luciferase Assay
Treatment vectors were transfected into 60% confluent HepG2, 293A, and H9C2 cells at
50,000 cells per well in twenty four well plates using the FUGENE Transfection kit (Roche,
Basel, Switzerland) following manufacturer’s instructions. Luciferase activity expressed by the
transfected cells was measured using the Promega Dual-Luciferase® Reporter Assay. A Renilla
luciferase vector was simultaneously transfected into the all the cells to form a baseline reading
for transfection efficiency. Expression was recorded in Relative Light Units (RLUs).
3.4 Enzyme-Linked Immunosorbent Assay (ELISA)

7. Ockunzzi 7
500 µL cell culture supernatant samples were collected from 24-well cell culture plates
with 50k cells in each, treated with either 1µg of the pT5SDF or pTSDF vector, or no treatment.
Analysis for stromal-cell-derived factor 1 (SDF1) protein levels for each group was conducted
according to Abcam® kit protocol. Data from the ELISA was obtained in duplicate.
3.5 Statistical Analysis
One-way ANOVA tests were used to compare means between treatment groups in all
Dual Luciferase and ELISAs, to confirm that the means are significantly different from one
another (p <0.05), significant statistical results are marked on graphs with an asterisk.
4. Results
In order to test SDF1 expression in liver cells, a treatment vector containing a tissue
specific promoter was cloned. The promoter region of the gene for thyroxine-binding globulin
(TBG) was selected for this purpose. TBG is a glycoprotein synthesized in the liver that bind and
transports the majority of thyroid hormone in circulation. TBG’s complete sequence and
transcriptional regulation has been laid out and the fragment -218 to +4 from the transcription
start site (TSS) demonstrated the greatest promoter functionality (Hyashi et al. 1993). The
sequence -435 to -26 bp within that region was utilized as the promoter, as tested by Yan et al.
(2012). To test the efficacy of TBG in hepatic cells (HepG2 cells, ATCC-HB-8065), it was tested
against the highly effective constitutive promoter for cytomegalovirus (CMV) (Magnusson et al.
2011). To optimize expression, and to test the effectiveness of enhancer elements, the expression
of non-enhancer containing vector treatments groups was compared with vector treatments
containing the Ru5 enhancer element from the replication region of the retroviral genome. Ru5,
when coupled with CMV has produced heightened gene expression following transfection
(Sundararaman et al. 2011). Luciferase was used as the reporter gene (Auld et al. 2008), and

8. Ockunzzi 8
expression was driven by combinations of the constitutive promoter, with and without the
addition of enhancer elements, enhancer elements alone, and liver-specific promoter TBG with
and without enhancer elements (Fig. 2A).
Both the TBG and CMV-driven vectors successfully expressed the reporter gene in
HepG2 cells, with CMV-driven vector expression, measured in relative light units (RLUs) being
on average 1.4614x102 times greater than their TBG-driven counterparts. Vector expression
driven by the TBG promoter was greater than enhancer-driven or Luciferase-driven expression.
HepG2 cells not treated with any vector were also assayed as a control group and showed no
expression. Additionally, vectors with the SDF-1 gene instead of the Luciferase reporter gene
showed no expression.
The pT5Luc and the pKCPLuc vectors, along with a negative control (Fig. 2A) were
transfected into HepG2 cells and Luciferase expression was tested 24 hours after transfection, 3
days after transfection (72hrs), and 10 days after transfection (Fig. 4). Control vectors showed no
expression at any time point. Both pT5L and pKLuc vector expression in HepG2 cells increased
close to tenfold (in RLUs) from the 24hr to the 3 day timepoints. At day 10 the pKCPLuc vector
expression was 0.4% of what it was at day 3, and at day 10 the pT5L vector expression was 8.1%
of what it had been at day 3. Both promoters show diminishing expression after 72 hours, but
expression due to the TBG promoter did not decrease as drastically as expression due to the
CMV promoter.
After the functionality of the TBG promoter was established in the hepatic cell line, its
specificity was tested when it was transfected into H9C2 and 293A cell lines to determine
expression levels in non-hepatocyte derived cell lines. The Luciferase reporter gene was then

9. Ockunzzi 9
replaced with the SDF1 gene and cloned into two vectors: one containing the TBG promoter and
Ru5 translational enhancer (Fig. 4), and the other only containing the TBG promoter- these were
then transfected into HepG2 cells. An enzyme-linked immunosorbent assay (ELISA) was
performed to determine the amount of SDF1 expressed and secreted by the transfected HepG2
cells.
The pT5Luc vector showed expression only in HepG2 cells, and the control vector
(PCR2.1) showed expression in H9C2 cells, but less than a half RLU, which could be due to
contamination of a well or a machine misreading. The hSDF-1 concentration in HepG2 cells
detected by the ELISA was 0.03866 pg/mL in cells transfected with 1µg DNA of pT5SDF
vector, and 0.0274pg/mL in cells transfected with 1µg DNA of pTSDF vector, at a mean
absorbance of 273nm (Fig. 6).
5. Discussion
Overexpression of the SDF-1 gene causes increased SDF-1 protein synthesis (Penn et al.
2010). The increased SDF-1 production elevated levels of CXCR4 (the receptor for SDF-1)
activation (Penn et al. 2009), which induces stem cell homing from the bone marrow (Lau and
Wang 2011) and differentiation into target cells in the tissue expressing the SDF-1 gene (Kollet
et al. 2003, Askari et al. 2003) Overexpression of SDF-1 has been achieved when vectors driven
by a strong promoter (alpha myosin heavy chain-αMHC and CMV) were transfected into target
cells (Sundararaman et al. 2011), these results replicated in this study with the use of a
cytomegalovirus (CMV) promoter (Fig. 3). Expression driven by a vector with a CMV promoter
would be sufficient to induce stem cell homing and thus conduct regenerative treatments,
however CMV is not tissue-specific and often requires viral treatment delivery (which could lead

10. Ockunzzi 10
to immuno-complications) or sometimes invasive procedures (i.e. surgical direct injections of
plasmid DNA) (Mali 2013).
Thyroxine binding globulin (TBG) is a protein synthesized by hepatocytes, and its
promoter region has been shown to successfully drive expression in a tissue-specific pattern
(Yan et al. 2012). Addition of the hepatic tissue-specific promoter sequence for TBG
demonstrated expression that was only 1/214 that of CMV-driven vector treatments (Fig. 3).
While the TBG vectors showed minimal comparative expression, they demonstrated specificity,
inducing luciferase expression only within HepG2 cells (Fig. 5). The TBG promoter’s specificity
allows for targeted treatments such as intravenous gene delivery, which has been achieved by
utilizing macrophages (white blood cells active in tissue repair) homing to infarct areas in
cerebral tissue (Tanaka et al. 2004). Delivery of the SDF-1 gene would cause production of the
SDF-1 protein in target cells, subsequent activation of CXCR4 receptors, initiating homing of
hematopoietic stem cells which have been shown to differentiate into hepatocytes (Lagasse et al.
2000).
TBG promoter expression showed less-diminishing expression than vectors delivered
using the CMV promoter between the 72 hour and 10 day timepoint. While initial expression of
CMV was high, it decreased more rapidly (as a percentage of the previous expression timepoint)
compared to TBG-driven expression (Fig. 4). If a 12 day timepoint had been taken, it is likely
that TBG would still be expressed in minute levels, while CMV would show no expression at all.
While TBG expression would still be low, it would still have the potential to induce stem cell
homing and continue to cause tissue rebuilding, potentially for a longer duration than CMV-
driven treatment vectors would. Prolonged SDF-1 expression in target tissues increases stem-cell
homing and tissue remodeling (Penn et al. 2009). Potential future experiments should retest

11. Ockunzzi 11
expression of pT5Luc and pKCPLuc vectors at the 24 hour, 72 hour, and 10 day timepoints, with
added timepoints of 12, 15, and 20 days.
Future experiments should also seek to test if expression levels as low as those of the
pT5Luc treatments would produce enough of an effect in live tissue for it to be a viable treatment
option. In previous studies, comparable levels of SDF-1 expression in the heart driven by vectors
containing the αMHC promoter were enough to induce stem cell homing and cardiac remodeling
(Sundararaman et al 2011). Testing if the TBG promoter could induce enough SDF-1 expression
to cause stem cell homing and tissue remodeling would involve controlled surgical damage to in
vivo specimens followed by direct injection of the plasmid into damaged tissue (as in Mali 2013,
Penn et al. 2009, Sandararaman et al. 2011, Kollet et al. 2003) and then measuring the extent of
tissue repair induced by the treatment. Additional studies could also test the efficacy of gene-
delivery options utilizing a TBG promoter, such as adenovirus vectors (Guzman et al. 1993).
Compared to other studies utilizing TBG promoter to drive transgene delivery
(Magnusson et al. 2011), the pT5Luc vector showed low expression. Reengineering new primers
as well as increasing transfection levels of treatments or adding a different enhancer region to the
plasmid could alleviate this issue and increase transgene expression. However, while overall
expression of the TBG-driven treatments was low compared to other transgene delivery vectors
(Magnusson et al. 2011, Zheng and Baum 2009) it still provides the opportunity for minimally
invasive, liver-specific regenerative treatments utilizing TBG as a tissue specific promoter.
6. Acknowledgments
I would like to thank Juventas Therapeutics and Penn Labs for giving me a position as a
research assistant, as well as Matthew Kiedrowski for overseeing my project and aiding me in

12. Ockunzzi 12
my studies. Additionally I would like to thank the Furman Advantage project for providing the
funding for my internship, as well as Dr. Rawlings for overseeing my Bio502 paper and project.

13. Ockunzzi 13
7. Figures and Legends
Panel A. Panel B. Panel C.
Figure 1. Gel electrophoresisconductedonhTBGPCR products(Lane 3, Panel A),pTLucsequentially
digested with BglII and HindIII-HF (Lane 3, Panel B), and pT5Luc sequentially digested with BglII and HindIII-HF
(Lane 1, Panel C), run on a 1% agarosegel next to a 1kbp DNA ladder to verify TBG presence (438bp).
~500bp
~500bp

14. Ockunzzi 14
Panel A.
Panel B.
Figure 2. Lineardemonstrationof insertsclonedintopCR2.1-hTBGplasmidthatwere transfected
intoHepG2 cells.Listof Vectornames inPanel A:A) pC5Luc, B) pT5Luc, C) pCLuc, D) pTLuc, E) p5Luc,
F) pLuc.The Luciferase reportergene wasdrivenbyacombinationof eithertissue-specificpromoter
Thyroxine-BindingGlobulin(TBG) orconstitutive promoterCytomegalovirus(CMV) withorwithout
Ru5 enhancerelements,andaBovine GrowthHormone (BGH) 3’ Poly-A tail. VectorsinPanel B:A)
pT5SDF, B) pTSDF.The Luciferase reportergene wasreplacedwiththe SDF-1gene invectors
transfectedintoHepG2cellsandan ELISA wasperformedonthese treatment groups(Fig6).
CMV Ru5 Luciferase BGH
TBG Ru5 Luciferase BGH
CMV Luciferase BGH
TBG Luciferase BGH
Ru5 Luciferase BGH
Luciferase BGH
A)
B)
C)
D)
E)
F)
TBG Ru5 SDF1 BGH
TBG SDF1 BGH
A)
B)

15. Ockunzzi 15
Figure 3. Luciferase expression24hrsaftertransfection(onalogscale) inHepG2 cells,with
relative lightunites (RLUs) shownbyplasmidconstructsfromFig.4alongwitha GFPvector and
a no-treatmentnegative control.Valuesin graphare meansof assaysperformedintriplicate.
Error barsindicate the SEM. A One-wayANOVAwasrunto determine if the meansof the
treatmentgroupswere significantlydifferentfromone another(p=0.000000654).
0.1
1
10
100
1000
10000
*pKCPLuc
*pCLuc
*p5Luc
*pLuc
*pT5Luc
*pTLuc
*pGFP3.1
*NT
*pTSDF1
*pT5SDF1
RelativeLightUnits(RLUs)
Transfected Vectors

16. Ockunzzi 16
Figure 4. Luciferase expressionof the vectorstransfectedintoHepG2cellsafter1,3, and 10
days,alongwithexpressionlevelsof a no-treatmentcontrol group atthose same timepoints.
Valuesingraphare meansof assaysperformedintriplicate,errorbarsindicate SEM. A One-way
ANOVA wasperformedondatafromeach timepointtoensure thatthe meanswere significantly
differentbetweentreatmentgroups(p=0.0000464, p=0.0000156, p=0.0000156).
0.1
1
10
100
1000
10000
1 3 10
RelativeLightUnits(RLUs)
Days After Transfection
pT5L Treatment
pKLuc Treatment
No Treatment
*
*
*
*
*
*
* * *

17. Ockunzzi 17
Figure 5. Luciferase expression24hrsaftertransfectioninHepG2,H9c2, and 293A cellstreated
withthe 1µg pT5Luc vector,accompaniedbya negative control- the PCR2.1vectorthatservesas
the original backbone forthe hTBG PCR insert.Errorbars indicate SEM. A One-wayANOVA was
run to determine if the meansweresignificantlydifferentbetweentreatmentgroups
(p=0.0000367).
0
0.5
1
1.5
2
2.5
3
HepG2 H9C2 293A
RelativeLightUnits(RLUs)
Cell Lines Transfected
pT5L Treatment
PCR2.1 - Control
*

18. Ockunzzi 18
Figure 6. Enzyme-linkedImmunosorbentAssay(ELISA)resultsdepictinghSDF-1protein
concentration(pg/mL) after24hrs in HepG2 cellstransfectedwith 1µgof pT5SDF1 or pTSDF
DNA,or no receivingnotreatment.Errorbarsindicate SEM. A One-wayANOVA wasrunto
determine if the meanswere significantlydifferentbetweentreatmentgroups (p=0.00000126).
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
*pT5SDF *pTSDF *NT
SDF-1Concentration(pg/mL)
Treatment Vectors

19. Ockunzzi 19
8. References
Amado LC, Saliaris AP, Schuleri KH, St. John M, Xie JS, Cattaneo S, Durand DJ, Kuang JQ,
Stewart G, Lehrke S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM. 2005. Cardiac
Repair with intramyocardial injection of allogenic mesenchymal stem cells after myocardial
infarction. PNAS 102(32):11474-11479.
American Liver Foundation [Internet]. 2015 July. New York (N Y): World Hepatitis Foundation;
[accessed 2016 Jan 2]. Available from:
http://www.liverfoundation.org/abouttheliver/info/progression/.
Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG,
Thomas JD, DiCorleto PE, Topol EJ, Penn MS. 2003. Effect of stromal-cell-derived factor 1
on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet
362(9385):697-703.
Auld DS, Thorne N, Nguyen DT, Inglese J. 2008. A specific mechanism for nonspecific
activation in reporter-gene assays. ACS Chem Biol 3(8):463-470.
BioGPS Gene Report: CXCL12 (chemokine (C-X-C motif) ligand 12). 2016. Jupier (FL): The
Scripps Research Institute (TSRI); [accessed 2016 Jan 2]. Available from:
http://biogps.org/#goto=genereport&id=6387.
Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. 2007. Culture of HepG2 liver cells on
three dimensional polystyrene scaffolds enhances cell structure and function during
toxicological challenge. J Anat 4:567-576.

20. Ockunzzi 20
Ekhterae D, Lin Z, Lundberg MS, Crow MT, Brosius FC, Nunez G. 1999. ARC inhibits
cytochrome release from mitochondria and protects against hypoxia-induced apoptosis in
heart-derived H9c2 cells. Circ Res 85(12):70-77.
Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. 1993. Efficient gene transfer into
myocardium by direct injection of adenovirus vectors. Circ res 73:1202-1207.
Hyashi Y, Mori Y, Janssen OE, Sunthornthepvarakul T, Weiss RE, Takeda K, Weinberg M, Seo
H, Bell GI, Refetoff S. 1993. Human thyroxine-binding globulin gene: complete sequence
and transcriptional regulation. Mol Endocrinol 7(8):1049-1060.
Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SW, Dabeva MD, Kahn J, Spiegel A, Dar
A, Samira S, Goichberg P, Kalinkovich A, Arenzana-Seisdedos F, Nagler A, Hardan I, Revel
M, Shafritz DA, Lapidot T. 2003. HGF, SDF-1, and MMP-9 are involved in stress-induced
human CD34+ stem cell recruitment to the liver. J Clin Invest 112(2):160-169.
Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M,
Weissman IL, Grompe M. 2000. Purified hematopoietic stem cells can differentiate into
hepatocytes in vivo. Nat Med 6(11):1229-1234.
Lau TT, Wang DA. 2011. Stromal cell-derived factor-1 (SDF-1): homing factor for engineered
regenerative medicine. Expert Opin on Biol Th 11(2):189-197.
Li J, Xue B, Yuan Y, Ma M, Zhu L, Milburn R, Le L, Hu P, Ye J. 2015. Construction of
recombinant human nerve growth factor eukaryotic vector and its expression in HEK293
cells. Chin J Biotech 31(3):411-420.

21. Ockunzzi 21
Magnusson T, Haase R, Schleef M, Wagner E, Ogris M. 2011. Sustained, high transgene
expression in liver with plasmid vectors using optimized promoter-enhancer combinations. J
Gene Med 13(7-8):382-391.
Mali S. 2013. Delivery systems for gene therapy. Ind J Hum Gen 19(1):3-8.
Penn MS. 2009. Importance of the SDF-1:CXCR4 Axis in Myocardial Repair. Am Heart Assoc.
104:1133-1135.
Penn MS. 2010. SDF-1:CXCR4 Axis if Fundamental for Tissue Preservation and Repair. Am J
Pathol 177(5):2166-2168.
Penn MS, Pastore J, Aras R. 2012. SDF-1 in myocardial repair. Gen Ther 19(6):583-587.
Pilarisetti K and Gupta SK. 2001. Cloning and Reliative Expression Analysis of Rat Stromal Cell
Derived Factor-1 (SDF-1): SDF-1 α mRNA is Selectively Induced in Rat Model of
Myocardial Infarction. Inflam 25(5):293-300.
Prockop DJ. 1997. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science
276(5309):71-74.
Rafi S, Lyden D. 2003. Therapeutic stem and progenitor cell transplantation for organ
vascularization and regeneration. Nat Med 9:702-712.
Sundararaman S, Miller TJ, Pastore JM, Kiedrowski M, Aras R, Penn MS. 2011. Plasmid-based
transient human stromal cell-derived factor-1 gene transfer improves cardiac function in
chronic heart failure. Gene Ther 18:867-873.

22. Ockunzzi 22
Tanaka S, Kitagawaw K, Sugiura S, Matuoka-Omura E, Sasaki T, Yagita Y, Hori M. 2004.
Infiltrating Macrophages as In Vivo Targets for Intravenous Gene Delivery in Cerebral
Infarction. Stroke 35:1968-1973.
Yan Z, Yan H, Ou H. 2012. Human thyroxine binding globulin (TBG) promoter directs efficient
and sustaining transgene expression in liver-specific pattern. Gene 506(2):289-294.
Zheng C, Baum BJ. 2009. Evaluation of promoters for use in tissue-specific gene delivery.
Methods in Mol Biol 434:205-219.