Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 4

Chapter 4. Receptor-mediated gene transfer


Chapter 4.
Preliminary studies on receptor-
mediated gene transfer using Vtg-
polylysine as DNA carrier and
identification of receptor-binding
domain in fish Vtg






137
Chapter 4. Receptor-mediated gene transfer
Abstract

The potential of receptor-mediated gene transfer using vitellogenin protein as DNA carrier
was explored. In these preliminary experiments, tilapia Vtgs were induced, purified and
labeled with
125
I. After injection, purified Vtgs could be preferably taken up by ovaries.
By modification with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), three types
of Vtg-poly-L-lysine conjugates were constructed and used in complexes preparation.
However, the efficiency of mediating DNA uptake from the Vtg-poly-L-lysine conjugates
by oocytes was not significantly higher than those by other tissues. Possible reasons for
this were discussed. Furthermore, recombinant Vtg fragments covering essentially the full
Vtg sequence were produced in E. coli and attempts of determining receptor-binding
domains were also made by in vivo binding assay, though inconclusive results were

observed.



138
Chapter 4. Receptor-mediated gene transfer
4.1. Introduction
Transgenic fish are not only an important experimental tool for developmental analyses of
gene expression and function, but also have enormous potential in aquaculture. In 1985,
Zhu et al. successfully made transgenic gold fish (Carassius auratus) through gene
transfer by microinjection. Since then, the production of transgenic fish has been reported
in many fish species and a variety of gene delivery methods have been developed and
successfully employed (Fletcher and Davies, 1991; Maclean and Rahman, 1994; Gong and
Hew, 1995; Chen et al., 1996). Common gene delivery methods used in transgenic fish
research include microinjection, electroporation, sperm carrier and particle bombardment.
Each method has its advantages and drawbacks. For example, microinjection is a popular
gene delivery approach used by many researchers but requires special equipment and
skilled personnel. Moreover, only a limited number of eggs can be injected at a time for
most fish species. In contrast, other gene delivery methods such as electroporation and
particle bombardment are more efficient in dealing with a large number of eggs but the
germ line integration rate of foreign genes is usually very low and special equipment is
also required. Thus, new gene delivery methods need to be developed that overcome these
deficiencies.

It is well known that receptor-mediated endocytosis (RME) provides a major pathway for
trafficking of extracellular molecules or ligands into animal cells. Based on the RME
process, a novel gene transfer method designated “receptor-mediated gene transfer” was
proposed (Wu and Wu, 1987). In their experiment, foreign DNA was transported into
hepatocytes by an asialoglycoprotein receptor-mediated pathway after administration of
complexes formed between DNA and asialoorosomucoid (ASOR)-poly-L-lysine (pLys)

139
Chapter 4. Receptor-mediated gene transfer
conjugates (Wu and Wu, 1987). The main advantage of the receptor-mediated gene
transfer is that it can be used to target specific cells in vivo after intravenous injection.
However, no attempts have been made to apply this gene delivery approach in fish.

In fish, Vtgs are synthesized in the liver under the control of E
2
and incorporated into
oocytes via receptor-mediated endocytosis (Selman and Wallace, 1982; Tyler et al., 1987).
Thus, Vtg is a candidate protein for a DNA carrier to use in transforming fish oocytes
through receptor-mediated gene delivery approach. If this gene delivery approach is
feasible in fish, foreign genes could be specifically introduced into the oocytes after
injection of the Vtg-DNA complexes into blood circulation of female fish and high
percentage of transgenic offspring would be expected. This gene delivery method is
effective, and does not depend on experienced personnel or special equipment.

In this study, receptor-mediated gene transfer will be tested in red tilapia (Oreochromis
mossambica). The reason for shifting the experimental model from zebrafish to tilapia is
that it is much easier to inject experimental materials through caudal artery of the tilapia
than of the zebrafish. Furthermore, the full-length cDNA sequence of tilapia (Oreochromis
aureus) vtg1 became available from GenBank during the project and it encodes a Vtg with
the homologous subdomains I-V (see Table 2-4 in Chapter 2). Thus, the tilapia vtg1 is a
potential orthologue of the zebrafish vtg2 and it also encodes a Vtg that is complete in
primary structure.

140
Chapter 4. Receptor-mediated gene transfer
4.2. Materials and Methods
4.2.1. E
2
induction and blood serum isolation
Female red tilapia (Oreochromis mossambica) (body weight 400 – 500g) were purchased
from a local fish farm and acclimated in a tank for two weeks. They were fed daily with
commercial fish food. E
2
stock solution was prepared as described in Chapter 3 (Section
3.2.1) and was injected intraperitoneally on days 1, 5, 9 and 14 at 4 µg E2/g body weight
according to Ding et al. (1989). Blood samples were collected through the caudal artery
from control and treated fish on days 4, 16 and 20, respectively, using pre-chilled
syringes. For serum isolation, blood was clotted on ice for 10-15 min prior to
centrifugation at 6000g for 10 min at 4 °C. Aprotinin (Sigma) was added to the serum at a
final concentration of 40 µg/ml and the fish serum was stored at – 70 °C prior to
purification.

4.2.2. Vtg purification by anion-exchange chromatography
Vtg was purified from tilapia serum by anion-exchange chromatography according to
Chan et al. (1991) with modifications. Briefly, to adjust the pH and ionic strength, buffer
A (50 mM Tris-HCl, pH 8.0) was added to the fish serum at a ratio of 3 : 1 (v/v). After
filtration, 1 ml of diluted serum sample was injected into an UNO Q-1 column (Bio-Rad)
integrated in a HPLC system (Pharmacia). Unbound proteins were washed away with 2.5
ml of buffer A (~ 2 times of bed volume) at a constant flow rate of 1 ml/min. For elution,
a gradient of 0 – 35% of buffer B (50 mM Tris-HCl, pH 8.0, 1 M NaCl) was applied over
15 ml, followed by holding at 35% of buffer B for 4 ml (see Fig. 4-3B). Elutes were
collected in 1 ml fraction and stored at – 80 ºC.

141
Chapter 4. Receptor-mediated gene transfer
4.2.3. Iodination of Vtg
Purified tilapia Vtg was labeled with
125
I using a solid phase oxidizing agent, 1,3,4,6-
tetrachloro-3α, 6α-diphenyl glycouril (Iodogen, Sigma) mediated method (Salacinski et
al., 1981). Briefly, an Iodogen tube was prepared by dispensing 100 µl of Iodogen solution
(0.1 mg/ml in chroloform) onto the bottom of a 12x75 mm glass tube, followed by
vacuum evaporation. A time course of radioiodination was determined based on the
method of Rudick (1998). Briefly, the Iodogen tube was rinsed with 1 ml of 50 mM Tris-
HCl (pH 8) to remove any loose flakes of Iodogen. Then, the following components were
added with gentle swirling: 90 µl of 50 mM Tris-HCl (pH 8.0), 10 µl of purified tilapia
Vtg (20 µg) and 0.5 µl of Na
125
I (50 µCi). Immediately, 4 µl of solution was removed and
spotted onto a nitrocellulose sheet. This process was repeated at 3 min intervals for a total
of 21 min. Finally, all nitrocellulose sheets were washed in 50 ml of washing buffer (25
mM Tris-HCl, 192 mM glycine, 12.5 mM NaI, 20% methanol, pH 8.3) and countered by a
Gammer counter (1470 Wizard, Wallac). Final labeling reaction was performed according
to the optimal duration determined and 20 µg of purified Vtg was radioiodinated at room
temperature. The
125
I-labeled Vtg was purification on a Sephadex G-25 column (PD-10,
Pharmacia Biotech) and stored at 4 °C prior to use.

4.2.4. Synthesis of Vtg-pLys conjugates
Purified tilapia Vtg was coupled to poly-L-lysine (pLys) through a disulfide bond after
modification by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)-
propionate (SPDP, Pierce) (Jung et al., 1981; Wagner et al., 1990). In this experiment,

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Chapter 4. Receptor-mediated gene transfer
























NH
2
Vtg

Vtg
HN-C-CH
2
-CH
2
-S-S-
O
poly-L-lysine
H
2
N
poly-L-lysineHS-CH
2
-CH
2
-C-NH
O
HN-C-CH
2
-CH
2
-S
O
poly-L-lysineS-CH
2
-CH
2
-C-NH
O
+ SPDP

+ SPDP
+
DTT
Vtg
Step 1 Step 2
Step 3
N
+
S
N
H
(pyridin -2-thione)




Fig. 4-1. Flow chart depicting the formation of Vtg-poly-L-lysine conjugates. Three steps
are included in the process, which is described in detail in Materials and Methods (Section
4.2.4). SPDP, N-succinimidyl 3-(2-pyridyldithio)-propionate; DTT, dithiothreitol.
(adapted from Jung et al., 1981; Wagner et al., 1990)

143
Chapter 4. Receptor-mediated gene transfer
two types of pLys with an average chain length of 36 (pLys
36
, MW 7500 Da, Sigma) and
144 lysine monomers (pLys
144
, MW 30,100 Da, Sigma) were used. Stock solutions of 3.33
nmol/µl for pLys

36
and 2 nmol/µl for pLys
144
were prepared in sodium phosphate buffer
(0.1 M sodium phosphate, pH 7.8, 0.1 M NaCl). The construction process included three
steps shown in Fig. 4-1. Results are summarized in Table 4-3.

Step 1. Modification of Vtg with 3-(2-pyridyldithio)-propionate by SPDP. First, a buffer
exchange with sodium phosphate was performed on a PD-10 column for the HPLC
purified Vtgs and the Vtg solution was concentrated using KwikSpin Micro Ultrafiltration
Units (30 kDa MWCO, Pierce). For modification of Vtgs used for type II conjugates (see
Table 4-3), 8 µl of SPDP stock solution (2 nmol/µl in 100% ethanol) was gradually added
to 430 µl of Vtg solution (0.86 nmol). The reaction mixture was vigorously mixed and
kept at 4 °C for 5 hr. Modified Vtgs were purified on PD-10 column with sodium
phosphate buffer. After an aliquot of modified Vtg was reduced by dithiothreitol (DTT),
the amount of dithiopyridine linkers per Vtg molecule was determined based on a molar
absorbance coefficient of 8.08 x 10
3
M
-1
• cm
-1
at 343 nm for the released product pyridin-
2-thione (Carlsson et al., 1978). For modification of Vtgs used for conjugates of types I
and III, the molar ratios between Vtg and SPDP were adjusted accordingly (see Table 4-3)
and the similar process was followed. SPDP modified Vtgs were stored at 4 °C before use.

Step 2. Modification of pLys with 3-mercaptopropionate by SPDP and DTT treatment.
First, for modification of pLys
36

by SPDP at a molar ratio of pLys
36
to SPDP of 1:1, 15 µl
of SPDP (20 nmol/µl) was mixed with 90 µl of pLys
36
(3.33 nmol/µl) and 83 µl of sodium

144
Chapter 4. Receptor-mediated gene transfer
phosphate buffer. The solution was vigorously mixed and kept at room temperature for 2.5
hr, followed by gel filtration on PD-10 column with sodium acetate buffer (20 mM sodium
acetate, pH 5.2, 0.1 M NaCl). For detection of the modified pLys, absorption at 211 nm
was measured for each elute. For modification of pLys
144
by SPDP at a molar ratio of
pLys
144
to SPDP of 1:2, 30 µl of SPDP (20 nmol/µl) was mixed with 150 µl of pLys
144
(2
nmol/µl) and the products were purified accordingly afterwards. Two standard curves
were prepared for quantification of pLys: 1) Y = 0.1035X-0.0061 for pLys
36
and 2) Y =
0.3633X + 0.0139 for pLys
144
(X: concentration of pLys in pmol/µl, Y: absorption at 211
nm). The amount of dithiopyridine linkers in modified pLys was determined as described
in step 1. Second, SPDP modified pLys

was reduced by DTT to form 3-
mercaptopropionate modified pLys. Briefly, 500 µl of SPDP modified pLys
36
(8.21 nmol)
or pLys
144
(3.01 nmol) was mixed with 12.5 µl of 1 M DTT and the solution was kept
under N
2
for 2 hr at room temperature. After gel filtration on PD-10 column with sodium
acetate buffer, the 3-mercaptopropionate modified pLys

was stored at – 20
°
C until use.

Step 3. Synthesis of Vtg-pLys conjugates. Three types of Vtg-pLys conjugates were
synthesized, Vtg-pLys
36
(type I), Vtg-pLys
144
(H) (type II) and Vtg-pLys
144
(L) (type III)
(see Table 4-3). Briefly, for making type I conjugates, 1 ml (1.95 nmol) of 3-(2-
pyridylditho)-propionate modified Vtg (with 4.9 linkers per molecule) was mixed with
100 µl (1.93 nmol) of 3-mercapto-propionate modified pLys
36
and the reaction was kept
under N

2
at 4 °C for ~20 hr. For making type II conjugates, the reaction was performed by
mixing 351 µl (0.8 nmol) of modified Vtg (with 10.1 linkers per molecule) with 175 µl
(0.832 nmol) of 3-mercaptopropionate modified pLys
144
. Similarly, for making type III

145
Chapter 4. Receptor-mediated gene transfer
conjugates, 467 µl (0.214 nmol) of modified Vtg (with 1.1 linker per molecule) was mixed
with 50 µl (0.238 nmol) of 3-mercapto-propionate modified pLys
144
. The Vtg-pLys
conjugates were separated from uncoupled 3-mercaptopropionate modified pLys by gel
filtration on a Bio-Gel P-100 column (with exclusion limit of 100 kDa) and eluted using
20 mM HEPES, pH 7.4, 0.15 M NaCl. The coupling degree was estimated based on the
increased OD
343
value as described above.

4.2.5. Formation of complexes between Vtg-pLys conjugates and DNA
Preparation of complexes between Vtg-pLys conjugates and DNA and gel retardation
assay were carried out according to Wagner et al. (1990, 1991). In order to form the
complexes, Vtg-pLys and DNA were directly mixed at a certain ratio in a buffer
containing 20 mM HEPES (pH 7.4) and 0.15 M NaCl, followed by incubation for 1 hr at
room temperature. The optimal ratio for neutralization between Vtg-pLys conjugates and
DNA was determined by gel retardation assay. Briefly, 1 µg of DNA (100-bp long, cut
from a plasmid by restriction enzymes) was labeled by [α-
32
P]dCTP using Nick

Translation Reagent Kit (BRL) according to the manufacturer’s protocol and purified by a
NICK Column (Pharmacia Biotech). A series of complexes were prepared between 0.45 µl
of
32
P-labeled DNA (~ 0.02 pmol) and increasing amount of Vtg-pLys conjugates. After
that, the complex mixture was loaded into a 1% agarose gel and resolved by gel
electrophoresis in 1x TAE buffer at 30 V for 2 hr. Finally, the agarose gel was dried and
autoradiography was performed at – 70
˚
C with Kodak's BioMax MS film.




146
Chapter 4. Receptor-mediated gene transfer
Table 4-1. Summary of primers used in amplification of eight tilapia vtg cDNA fragments
by RT-PCR


Amplified
fragment

Primer name

Primer sequence
†
Position in
tilapia vtg1
CDS

‡
1F 5'-CCGGATCCGACCAGTCCAACTTGGCCC-3'
BamH I
46-64 LVIa
1R 5'-CCGAATTCTCTCTAGCGACAGCCTCC-3'
EcoR I
702-720
2F 5'-CCGGATCCCTGCAGTATGAG-3'
BamH I
846-864 LVIb
2R 5'-CCGAATTCACAGCATCTCTGA-3'
EcoR I
2291-2309
3F 5'-CCGGATCCTCTGTGCTGTCTGGTTATG-3'
BamH I
2308-2326 LVIc
3R 5'-CCGAATTC
TTGAGACCAGGTGCCA-3'
EcoR I
3239-3257
4F 5'-CCGGATCCGGAGATAAAGCAGCAGAA-3'
BamH I
3138-3156 PV
4R 5'-CCGAATTCCTGGCAGTTTGTCCCA -3'
EcoR I
4246-4264
5F 5'-CCGGATCCGTCGCTGAGAAGGACAACT-3'
BamH I
4078-4096 LVII

5R

5'-CCGAATTCAAGCACACTGAGGAGTGCAG-3'
EcoR I
5346-5365

2aF 5’-CCAGATCTTTCTCACCTTTCAACATTTTG-3’
Bgl II
743-763 LVIb1
2aR 5’-CCGAATTC
GAACCTGGAAATTACAGTG-3’
EcoR I
1333-1351
2bF 5’-CCGGATCCGCTGAGAACCACAGAGTG-3’
BamH I
1271-1288 LVIb2
2bR 5’-CCGAATTC
TGCTGCAGCAACAGAAAC-3’
EcoR I
1820-1837
2cF 5’-CCGGATCCTACATGAAGGCCATGGCC-3’
BamH I
1781-1798 LVIb3
2cR 5’-CCGAATTC
ATCTCTGAATACGTTTCTC-3’
EcoR I
2293-2311
†Overlapping sequence with tilapia (Oreochromis aureus) vtg1 cDNA is in italic letters.
For primer locations, see Fig. 4-7.

‡Tilapia (Oreochromis aureus) vtg1 cDNA sequence is from GenBank (accession No.
AF017250). CDS, coding sequence.

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Chapter 4. Receptor-mediated gene transfer
4.2.6. Amplification of eight tilapia vtg cDNA fragments by RT-PCR
Red tilapia (Oreochromis mossambica) liver total RNA was extracted using TRIzol
Reagent (GIBCO BRL) according to the manufacturer’s instructions. Eight pairs of primer
with BamH I (or Bgl II) and EcoR I linkers were designed based on tilapia (Oreochromis
aureus) vtg1 cDNA sequence (GenBank accession No. AF017250) and used in
amplification of eight tilapia vtg cDNA fragments by RT-PCR (Table 4-1). RT-PCR was
performed using Access RT-PCR System (Promega). Briefly, first strand vtg cDNAs were
synthesized using AMV reverse transcriptase at 48°C for 45 min. Inactivation of the
reverse transcriptase and denaturation of mRNA/cDNA were performed at 94°C for 2 min,
followed by 35 to 40 cycles of PCR for amplification of target sequences using the
following conditions: 94 °C for 30 sec, 55~67 °C for 1 min, 72 °C for 2 min, final
extension at 72 °C for 8 min. Amplified products were resolved in 1% agarose gel and
bands with anticipated sizes were cut under UV illumination. vtg cDNA fragments were
recovered from the gel using QIAquick Gel Extraction Kit (QIAGEN).

4.2.7. Construction of GST-Vtg fusion protein expression vectors
Glutathione S-transferase (GST)-Vtg fusion protein expression vectors were constructed
based on an expression vector pGEX-2TK (Pharmacia Biotech, Fig. 4-2A). Briefly, the
expression vector pGEX-2TK was linearized by restriction enzyme digestion with BamH I
and EcoR I, and subsequently ligated with each of the following five vtg cDNA fragments,
LVIa, LVIb, LVIc, PV and LVII, which were also digested by BamH I and EcoR I. Ligation
was performed at 14 °C overnight in the presence of T4 DNA ligase (GIBCO). The


148

Chapter 4. Receptor-mediated gene transfer

pGEX-2TK
A
pRSET
2.9 kb
A
B
C

Protein kinase site



P
T7


RBS ATG 6xHis Xpress Epitope EK
CGTCGTGCATCTGTT
vtg fragment
Stop

Fig. 4-2. Maps of original and modified expression vectors used in expression of
recombinant Vtg fragments. A: Vector map of pGEX-2TK. Arrows indicate cloning sites
for vtg cDNA insert. B: Vector map of pRSET-A (original). Arrows indicate cloning sites
for a new cDNA fragment LVIa’, which was amplified by PCR using a 48 mer forward
primer (for sequence, see Materials and Methods, Section 4.2.8) and the reverse primer 1R
(Table 4-1) from the plasmid pGST-LVIa. C: Partial map of the modified pRSET-A
vector (pRSET’), showing an extra protein kinase site (arrow) located between the six

histidine residues and vtg cDNA insert. Restriction sites flanking the vtg insert are shown.

149
Chapter 4. Receptor-mediated gene transfer
resulting expression vectors were named as pGST-LVIa, pGST-LVIb, pGST-LVIc,
pGST-PV and pGST-LVII.

4.2.8. Construction of 6xHis-tagged Vtg expression vectors
The original 6xHis expression vector pRSET A (Invitrogen, Fig. 4-2B) was first modified
with an insertion of a protein kinase recognition site coding sequence to facilitate the
labeling of 6xHis-tagged Vtgs by [γ-
32
P]ATP. Briefly, a 48-mer forward primer (5'-
CCAGATCTCGTCGTGCATCTGTT-GGATCCGACCAGTCCAACTTGGCCC-3') was
designed which contains (5’ to 3’) a Bgl II cutting site (AGATCT), a sequence
(CGTCGTGCATCTGTT) encoding a protein kinase recognition motif (RRASV) and the
sequence of primer 1F (excluding the two protection nucleotides at 5’ end), which
contains a BamH I site. PCR was performed using this 48-mer primer and the reverse
primer 1R in order to amplify a fragment (LVIa’) from the plasmid pGST-LVIa. After
restriction enzyme digestion with Bgl II and EcoR I, the amplified fragment was ligated
with the pRSET A vector (linearized by BamH I and EcoR I), resulting in a modified
vector, named as pRSET'-LVIa, which encodes a 6xHis-tagged LVIa with a protein kinase
recognition motif (Fig. 4-2C). For construction of other 6xHis-Vtg expression vectors, the
pRSET'-LVIa was cut by BamH I and EcoR I, followed by ligation with a respective vtg
cDNA fragment, which was obtained either by restriction enzyme digestion of construct
pGST-LVIb, pGST-LVIc or pGST-LVII (by BamH I and EcoR I), or by RT-PCR
amplification for cDNA fragment LVIb1, LVIb2 or LVIb3. Thus, seven 6xHis-Vtg
expression vectors were constructed and named as pRSET'-LVIa, pRSET'-LVIb, pRSET'-
LVIc, pRSET'-LVII, pRSET'-LVIb1, pRSET'-LVIb2 and pRSET'-LVIb3.

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Chapter 4. Receptor-mediated gene transfer
4.2.9. Expression and purification of recombinant vtg fragments
Recombinant proteins of GST-Vtgs and 6xHis-Vtgs were expressed in E. coli BL21 cells
at 28
˚
C after induction with 0.1-1.0 mM (final concentration) of isopropyl-1-thio-β-D-
galactopyranoside (IPTG, Sigma). Bacteria were lysed with lysozyme and Triton X-100
according to Gong and Hew (1994). Briefly, after centrifugation, the bacterial pellet was
re-suspended in lysis buffer at a ratio of bacteria to lysis buffer of 1 : 2 (w: v) followed by
addition of lysozyme to a final concentration of 2.5 mg/ml. The lysis buffer contained
15% (w/v) sucrose, 2 mM EDTA, 5 µg/ml aprotinin, 1 mM PMSF, 1 mM DTT and 50
mM Tris-HCl (pH 8) (GST-Vtgs) or 15% (w/v) sucrose, 5 µg/ml aprotinin, 1 mM PMSF,
500 mM NaCl and 50 mM sodium phosphate buffer (pH 7) (6xHis-Vtgs). After mixing,
Triton X-100 was added to a final concentration of 1% (v/v) and the suspension was
mixed vigorously. Then, an equal volume of lysis buffer was added and the solution was
supplemented with MgCl
2
and Dnase I (final concentrations of 5 mM and 20 – 40 µg/ml,
respectively). Finally, after centrifugation at 15000 rpm, 4 °C for 15 min, the supernatant
containing soluble recombinant proteins was removed and stored at 4 °C .

Purification of recombinant Vtgs was carried out using either GST Sepharose 4B
(Pharmacia Biotech) for GST-Vtgs or Talon Metal Affinity Resins (Clontech) for 6xHis-
Vtgs according to the manufacturers’ protocols. Briefly, 50% bead slurry was added to the
clear supernatant of bacteria lysate at a ratio of 1 : 20 (v/v), followed by incubation at 4 °C
for 30 – 60 min on a rocking platform. Then, beads were spun down at 700 g, 4 °C for 5
min and washed with cold washing buffer (100 mM NaCl, 0.5% NP-40, 1 mM DTT in 20
mM Tris-HCl, pH 8 for washing GST fusions; 500 mM NaCl, 25 mM Imidazole, 0.5%

151
Chapter 4. Receptor-mediated gene transfer
NP-40 in 50 mM sodium phosphate buffer, pH 7 for washing 6xHis-Vtgs) three times with
10 min each at 4 °C on a rocking platform. Finally, elution buffer (containing 10 mM
reduced GSH in 50 mM Tris-HCl, pH 8 for GST-Vtgs or 300 mM NaCl, 150 mM
Imidazole in 50 mM sodium phosphate buffer, pH 7 for 6xHis-Vtgs) was added to the
beads, followed by incubation at room temperature for 5 min. After centrifugation, the
supernatant containing purified recombinant Vtgs was collected and kept at 4 °C for
further analysis.

4.2.10. Labeling of 6xHis-Vtgs by [γ-
32
P]ATP
6xHis-Vtgs were labeled by [γ-
32
P]ATP on beads according to the manufacturer’s
protocol (Pharmacia Biotech). Briefly, recombinant protein bound beads were washed in 1
x heart muscle kinase buffer (HMK) (20 mM Tris, pH 7.5, 0.1 M NaCl, 12 mM MgCl
2
),
spun down and kept on ice. To 20 µl of beads, 30 µl of protein kinase reaction mixture [3
µl of 10 x HMK, 3 µl of 10U/µl bovine heart kinase (dissolved in 1 x HMK buffer), 3 µl
of [γ-
32
P]ATP (3000 Ci/mmol) and 21 µl of dH
2
O] was added. Then, the solution was
mixed by gentle agitation and incubated at 4 °C for 30 min. After that, the beads were
washed twice in ice-cold 1 x PBS and spun down. Finally, radio-labeled recombinant Vtgs

were eluted from the beads and stored at 4 °C until use. GST was also labeled by [γ-
32
P]ATP as a control.

4.2.11. Polyacrylamide gel electrophoresis
One-dimensional discontinuous sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was carried out using a Bio-Rad minigel apparatus according to Celis and

152
Chapter 4. Receptor-mediated gene transfer
Olsen (1998). The composition of the gels were 5% (w/v) acrylamide-bisacrylamide
(Acr/Bis) mixture (C=3.3), 0.125 M Tris-HCl (pH 6.8) and 0.1% SDS (for stacking gel);
8-10% (w/v) Acr/Bis, 0.374 M Tris-HCl (pH 8.8) and 0.1% SDS (for separating gel).
Running buffer contained 25 mM Tris-HCl (pH 8.3), 0.192 M glycine and 0.1% SDS, and
the sample buffer contains 80 mM Tris-HCl (pH 6.8), 2% SDS, 7.5% (v/v) glycerol and
5% 2-mercaptoethanol (v/v). For resolving
125
I-labeled native Vtgs, nondenaturing
polyacrylamide gel electrophoresis (NPAGE) was performed according to Safer (1998).
Briefly, the composition of the gels were 3% (w/v) Acr/Bis and 0.124 M Tris-HCl (pH
6.7) for the stacking gel; 5.5% (w/v) Acr/Bis and 0.374 M Tris-HCl (pH 8.9) for the
separating gel. Running buffer was composed of 50 mM Tris-HCl (pH 8.3) and 0.384 M
glycine; sample buffer composed of 62 mM Tris-HCl (pH 6.8) and 10% glycerol (v/v).
Samples were either denatured at 95
º
C for 5 min prior to loading (for SDS-PAGE) or
directly loaded without denaturation (for NPAGE). Gel electrophoresis was performed
with constant voltage at 50 V for 0.5 h, followed by 100 V for 2 h. Proteins were stained
with 0.1% (w/v) Coomassie Brilliant Blue R-250 (BDH) which was dissolved in 25%
methanol and 10% acetic acid. Protein quantification was performed based on Bradford

colorimetric assay method using Bio-Rad Protein Assay Kit (standard or microassay
procedure) with BSA (Sigma) as a standard.

4.2.12. Western blot
Western blot was performed according to the manufacturer’s protocol (Pierce). Briefly,
after SDS-PAGE, resolved proteins were transferred onto a piece of nitrocellulose paper
using Bio-Rad Mini Trans-Blot Cell at 250 mA for 2.5 hr. The transfer buffer contained
20 mM Tris base and 150 mM glycine. After blocking in 50 ml of blocking buffer (25 mM

153
Chapter 4. Receptor-mediated gene transfer
Tris-HCl, pH 8.0, 50 mM NaCl, 50 mg/ml BSA) at 4
º
C overnight, the nitrocellulose
membrane was incubated in 10 ml of blocking buffer with 1 : 5000 diluted Anti-HisG
Antibody (1 mg/ml, Invitrogen) for 1 hr at room temperature with shaking. Then, the
membrane was washed 3 times in washing buffer (25 mM Tris-HCl, pH 8.0, 50 mM NaCl,
5 mg/ml BSA) for 10 min each, followed by incubation in 1 : 2000 diluted horseradish
peroxidase conjugated goat anti-mouse IgG secondary antibody (0.8 mg/ml, Pierce) for 1
hr at room temperature. After final washes, SuperSignal West Pico Chemiluminescent
Substrate (Pierce) was added to the membrane. Signals were captured by exposure with a
piece of Hyperfilm ECL (Amersham pharmacia biotech).

4.2.13. Administration method, sampling criteria and sample treatment
Red tilapia (Oreochromis mossambica) (~ 10 cm in body length) was administrated
radioisotope labeled proteins or DNAs by injection into the caudal artery. The injected
materials included
125
I-labedled native tilapia Vtgs (~ 40,000 cpm/fish),
32

P-labeled 100
bp DNA (~ 200,000 cpm/fish), complexes of
32
P-labeled DNA and Vtg-pLys conjugates
(~ 200,000 cpm/fish) and
32
P-labeled recombinant Vtg fragments (~250,000 cpm/fish).

To evaluate the developmental stages of oocytes, ovaries from experimental fish were
removed and weighed. The diameter of randomly selected oocytes was measured prior to
further processing of the ovary samples. The gonadal index (GI) was calculated according
to the following formula GI = (weight of ovary/weight of fish body) x 100%. Female fish
bearing oocytes at developmental stages between vitellogenic (oocyte diameter = ~ 0.7
mm) and preovulatory stages (oocyte diameter = ~ 2 x 3 mm) (Kraft and Peter, 1963;

154
Chapter 4. Receptor-mediated gene transfer
Quek, 1985) were used for further analysis. Experimental fish bearing oocytes out of the
above ranges were discarded accordingly.

Seven tissues were isolated, including the ovary, liver, gut, gill, heart, spleen and kidney.
The radioactivities in those tissues were either measured directly by a gamma counter
(1470 Wizard, Wallac) for
125
I or measured after treatment by a liquid scintillation counter
(1414 Guardian, Wallac) for
32
P.
32
P samples were pre-treated with a tissue solubilizer

NNCS502 (Amersham) at 10 ml/g tissue overnight before counting. For
125
I-injected fish,
the collective radioactivity in the remaining parts of fish was calculated by summing the
radioactivities from all remaining tissues and labeled as “remains”. For
32
P injected fish,
the collective radioactivity in the remaining parts of fish (labeled as “others”) was
obtained by measuring the radioactivities of portions of the remaining tissues and then
multiplying accordingly based on the proportion of the sampled tissues in the remaining
parts of fish (by weight). The “radioactivity recovery rate” was defined as the total
radioactivity of whole fish divided by the injected radioactivity, and the “relative
radioactivity” was defined as the radioactivity of each tissue divided by the total
radioactivity of whole fish.



155
Chapter 4. Receptor-mediated gene transfer
4.3. Results and Discussion
4.3.1. Purification of tilapia vitellogenin proteins
To obtain a sufficient amount of native vitellogenin for construction of Vtg-polylysine
conjugates, female red tilapia were injected with E
2
. After injection, serum proteins were
examined by SDS gel electrophoresis. We found that multiple Vtg proteins were induced
by E
2
treatment, especially after the 4
th

injection, as revealed by SDS-PAGE analysis (Fig.
4-3A). As shown in Fig. 4-3A, two proteins with molecular weights of ~190 and ~130
kDa were greatly enhanced in female fish after the 4
th
E
2
injection compared with those
from the control females and females after the 1
st
E
2
injection. In serum of male fish, both
bands were absent (Fig. 4-3A, lane 1). It was reported that two Vtg subunits of 180 and
130 kDa were induced in male tilapia (Oreochromis aureus) after E
2
treatment (Ding et
al., 1989). Similarly, in male tilapia (O. mossambicus), the molecular weights of two E
2

induced Vtg subunits were reported as 200 and 130 kDa, respectively (Kishida and
Specker, 1993). Thus, the enhanced proteins of ~190 and ~130 kDa in this experiment
were assumed to be two Vtg subunits.

Based on a procedure used in purifying Vtgs from nile tilapia (O. niloticus) (Chan et al.,
1991), tilapia (Oreochromis mossambica) Vtgs were purified by HPLC from serum
proteins of E
2
treated female fish. As shown in Fig. 4-3B, a potential Vtg peak was
identified in the gel filtration profile when the concentration of NaCl reached 0.35 M in
the elution buffer. After SDS-PAGE analysis, it was found that the potential Vtg peak was

mainly composed of the Vtg subunit of ~190 kDa (Fig. 4-3A, lane 6).

156
Chapter 4. Receptor-mediated gene transfer
A
B
M
1
2 3 4 5678
198
113
75
48.9
33.1
kDa
*
*
9
23 456789101 1112 13 14 15
16
17 18 19 2120 22
0.5
1.0
1.5
A
280
Gradient %B
35
0
Tube number

23 24
~500 kDa


Fig. 4-3. Gel electrophoresis and HPLC purification profile of tilapia serum proteins. A:
SDS-PAGE analysis of tilapia serum proteins (lanes 1-4) and HPLC fractions (lanes 5-8).
NPAGE analysis of
125
I-labeled Vtg is shown in lane 9. Lanes 1, 2, 3 and 4 contain serum
proteins from control male, control female, female after first injection with E
2
and female
after fourth injection with E
2
, respectively (0.5 µg protein/lane). Lanes 5, 6, 7 and 8
contain HPLC fractions 18, 19, 20 and 21 (in B), respectively (20 µl fraction/lane). Two
Vtg subunits of ~190 and ~130 kDa are marked by arrowheads and asterisks, respectively.
B: HPLC profile of serum proteins from female tilapia after first injection with E
2
. An
arrow indicates a potential Vtg peak.

157
Chapter 4. Receptor-mediated gene transfer
4.3.2. Purified native vitellogenins were preferably taken up by ovaries
To test whether the HPLC purified tilapia Vtgs can be recognized by their receptors and
subsequently taken up by fish oocytes, tilapia Vtgs were labeled with
125
I, and injected
into tilapia caudal artery. Based on the radioiodination time course, Vtg proteins were

labeled with Na
125
I by the Iodogen mediated method for 15 min at room temperature,
resulting in a 82% incorporation rate of Na
125
I. The integrity of
125
I-labeled Vtgs was
monitored by nondenaturing polyacrylamide gel electrophoresis (NPAGE) analysis. As
shown in Fig. 4-3A (lane 9), no apparent degradation was observed after radioiodination.

After injection of
125
I-labeled Vtgs (~ 40000 cpm/fish) into female fish, various tissues
were isolated at different time points and the radioactivities of certain tissues and the
remains of fish were measured using a gamma counter. The mean radioactivity recovery
rates ranged from 43.4% to 58.0% at different time points, indicating there was no
significant difference in the radioactivity recovery rates between different time points
(Table 4-2). Essentially, nearly half of the injected radioactivity was lost at 12 hr after
injection and probably part of it was lost during the injection. Thus, for subsequent assays,
relative radioactivity was used, which was defined as the radioactivity of each tissue
divided by the total radioactivity of whole fish. As shown in Table 4-2 and Fig. 4-4, 12 hr
after injection, more than half of the total radioactivity of whole fish (52.4%) was detected
in the remaining tissues of fish, while only 19.7% of the total radioactivity was
accumulated in the ovary. However, with the time lapsed, the relative radioactivity in the
ovary increased to 45.5%, 52.0% and 68.1% at 24, 48 and 72 hr respectively after
injection. In contrast, the relative radioactivities in essentially all other tissues decreased

158
Chapter 4. Receptor-mediated gene transfer

Table 4-2. Relative radioactivities in seven tissues and the remains of fish examined at 12,
24, 48 and 72 hr after injection with
125
I-labeled Vtg.

Time
Tissue
12 hr
†

(n = 3)
24 hr
(n = 5)
48 hr
(n = 4)
72 hr
(n = 4)
Ovary 19.7 ± 11.3
‡
45.5 ± 4.0 52.0 ± 28.6 68.1 ± 6.1
Liver 14.1 ± 5.3 10.9 ± 4.8 5.5 ± 2.8 7.3 ± 4.9
Gut 5.3 ± 4.1 6.1 ± 3.1 4.2 ± 2.5 1.5 ± 0.3
Gill 6.6 ± 0.4 6.2 ± 0.3 5.8 ± 2.7 8.4 ± 4.5
Heart 1.1 ± 1.1 0.6 ± 0.7 0.2 ± 0.04 0.2 ± 0.1
Spleen 0.3 ± 0.2 0.4 ± 0.2 0.3 ± 0.2 0.3 ± 0.1
Kidney 0.3 ± 0.1 0.3 ± 0.2 0.3 ± 0.1 0.2 ± 0.1
Remains 52.4 ± 14.3 30.0 ± 4.7 31.8 ± 23.0 14.2 ± 1.1
Radioactivity
recovery rate
50.6 ± 4.0 54.1 ± 11.7 43.4 ± 11.5 58.0 ± 13.8

†The mean gonad indexes of fish in experimental groups of 12, 24, 48 and 72 hr were 4.67
± 0.16 %, 4.16 ± 2.67 %, 4.69 ± 1.74 % and 3.68 ± 1.92 % (mean ± SD%), respectively.
‡Mean ± SD (%).


159
Chapter 4. Receptor-mediated gene transfer
0
10
20
30
40
50
60
70
80
90
ovary liver gut gill heart spleen kidney others
% of total radioactivity of whole fish
12 hr
24 hr
48 hr
72 hr
remains


Fig. 4-4. Relative radioactivities in seven tissues and the remains of fish examined at 12,
24, 48 and 72 hr after injection with
125

I-labeled tilapia vitellogenin proteins (~ 40000
cpm/fish). Relative radioactivity is defined as radioactivity of each tissue divided by the
total radioactivity of whole fish and is presented as mean ± SD %. For information about
the number of fish examined and their mean gonad index, see legend of Table 4-2.

160
Chapter 4. Receptor-mediated gene transfer
from 12 to 72 hr after injection, indicating that the injected
125
I-Vtgs were gradually
transported into the ovary through blood (Fig. 4-4). Thus, exogenously introduced native
tilapia Vtgs can be predominantly, if not specifically, taken up by vitellogenic ovaries.

It has been reported that the number and affinity of ovarian vitellogenin receptors in
tilapia (Oreochromis niloticus) change during oocyte growth with an increase of receptor
number from previtellogenic to vitellogenic stages and remains constant thereafter (Chan
et al., 1991). Similar trends were also observed in rainbow trout (Salmo gairdneri) (Tyler
et al., 1988b). Thus, it is critical to choose fish bearing ovaries at similar developmental
stages to make reasonable comparisons between different injection groups. As listed in the
legend of Table 4-2, the mean gonad indexes (GI) of fish in the four experimental groups
were similar (3.68–4.69%), which validated the comparison. In addition, the diameter of
oocytes in these groups of fish ranged between 0.7 to 2 x 3 mm, indicating the majority of
oocytes were between vitellogenic to preovulatory stages.

Thus, the injected Vtgs were supposed to be uptaken by oocytes through receptor-
mediated endocytosis process. However, detailed studies regarding the distribution of
injected Vtgs inside the oocytes need to be carried out in the future to further confirm this.

4.3.3. Synthesis of Vtg-poly-L-lysine conjugates
Vtg-poly-L-lysine conjugates were synthesized using HPLC purified tilapia vitellogenin

and poly-L-lysine (pLys) through ligation by disulfide bonds as depicted in Fig. 4-1. To
illustrate the whole process, the synthesis of type II conjugate Vtg-pLys
144
(H) was
described below. First, dithiopyridine groups were introduced into both Vtg and pLys
144


161