# A Sensitive and Rapid Assay for Investigating Vertical Transmission of Hepatitis B Virus via Male Germ Line Using EGFP Vector as Reporter

Authors: Mohamed Morsi M. Ahmed; Tian-Hua Huang; Qing-Dong Xie
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virusS gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed.


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## Body

## 1. Introduction

Hepatitis B virus (HBV) is a DNA virus, replicating almost exclusively in the
liver; see [1]. Although effective recombinant vaccines are available, HBV
infection is still a major global health problem and each year, acute and
chronic HBV infection causes about 1 million deaths [2]. Hepatitis B is a worldwide public health
problem. WHO estimates that approximately 350 million people are chronically
infected with hepatitis B virus (HBV), and that the prevalence is more than 20%
in certain highly endemic area of Africa and Asia (EPI NEWS, No. 25, 2002). Sequelae of HBV infection are severe. Up to 15% of carriers eventually develop
primary hepatic carcinoma; the younger a patient is when they acquire chronic
infection, the higher the risk of primary hepatocellular carcinoma. Premature
mortality from chronic liver disease occurs in 15–25%. Therefore, studies on transmission of HBV are of
substantial importance in virology as well as in public health.The transmission routes of HBV through blood transfusion [3, 4]; body
fluids including serum, saliva, vaginal secretions, breast milk, and semen [5, 6]; intrauterine infection [7, 8]; cell, tissue, and organ transplantation [9, 10] and others including hemodialysis units and intravenous drug
injection [11, 12] have been
documented. The known transmission of HBV does not constitute a menace
to man because we are able to identify effective preventive measures. The unknown transmissions have greater risk potential because we do not know where they are
when HBV attacks us. In 1985, Hadchouel et al. [13] confirmed the presence of integrated HBV DNA sequences in spermatozoa from two of three
patients with HBV infection using the molecular hybridization. They
assumed that the presence of integrated sequences in spermatozoa suggested the
possibility of true vertical transmission of HBV via germ line. Nobody confirmed
their assumption in more than ten years, because neither experimental animals
nor cell culture systems had been available. Study on vertical
transmission of HBV genes using embryos produced from human oocytes fertilized with human spermatozoa
carrying HBV genes would be an ideal model, but such a system presents major logistical,
moral, ethical, and methodological problems. Thus, it is crucial to
establish a proper assay for such study.Spermatozoa of a wide
variety of species such as mouse, guinea pig, and human can fuse with zona-free
hamster oocytes. Interspecific in vitro fertilization has been widely used by
investigators for studying on morphological and molecular details of sperm-egg
interactions [14, 15]. Huang et al. employed this method for investigating
transmission of HBV via male germ line and avoided the mentioned moral and ethical problems.
They firstly provided the direct evidence that HBV DNA integrated into human sperm chromosomes and showed that human sperm carrying
HBV genes can pass through oolemma to enter into the oocyte and complete
fertilization normally [16]. Other investigators demonstrated that after fertilization the sperm-mediated
HBV genes are able to replicate themselves and express their functions at mRNA
and protein level in early embryonic cells [17–19]. But it was the
sample-consuming, time-consuming, and energy-consuming to utilize the mentioned
experimental model because not all sperm either from the semen of patients with
HBV chronic infection or from the human spermatozoa exposed in vitro to the recombinant plasmid pBR322-HBV were integrated by HBV DNA.
After fertilization it was difficult to see which embryo contained HBV genes delivered by human sperm. To obtain the mentioned results, many embryos as the
test materials were needed and
the experiment had to be repeated many times in the previous studies.Enhanced
green fluorescent protein (EGFP) as a good reporter has been widely used for
biological and medical research. In the present study we constructed a
recombinant plasmid pIRES2-EGFP-HBs containing HBs and EGFP
genes, which enable us to distinguish between the embryos with and without HBs gene through observation of the
presence of green fluorescence. The aim of this study was to provide a
rapid and sensitive assay
for exploring feasibility of vertical transmission of HBV and other viruses via
the germ line.


## 2. Materials and Methods

All experiments of the present work were performed under the approval of the Internal Review Board of Shantou University Medical College.


### 2.1. Materials

Biggers-Whitten-Whittingham (BWW) medium supplemented with 0.3% bovine
serum albumen (BSA, Sigma Chemicals Co., ST. Louis , MO) was
prepared for human sperm preparation, oocyte collection, insemination and
subsequent handling. Ovum culture medium (OCM, from Flow Laboratories, Germany)
containing 10% heat-inactivated fetal bovine serum was used in the post-insemination
culture.


### 2.2. Methods

#### 2.2.1. Construction of Recombinant Plasmid pIRES2-EGFP-HBs

The expression vector for
HBs and EGFP was constructed as Figure1.Figure 1
Diagram of constructed recombinant plasmid pIRES2-EGFP-HBs.The HBs gene was amplified (403 bp) from plasmid pBR322-HBV by PCR and subcloned to plasmid pIRES2-EGFP to construct recombinant plasmid
pIRES2-EGFP-HBs. The PCR mixture consisted of 5μL 10X Taq buffer, 4 μL of
2.5 mM of MgCL2, 200 μmol/L of
each deoxynucleoside triphosphate (Invitrogen, Guangzhou, China), 20 pmol of each primer with EcoR I and
Sal I restriction sites (Takara Biotech, Japan) including forward: 5′-CGGAATTCTATCGCTGGATGTGTCTGC-3′ and reverse: 5′-GGTCGACAGACTTGGCCCCCAATAC-3′,
2.5 U Taq polymerase  and sterile ddH2O in
a final volume of 50 μL. The amplification program was as follows: denaturing
at 94°C for 5 minutes, and 35 cycles each at 94°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 45 seconds,
followed by a final extension at 72°C for 10 minutes. The amplification products were routinely analyzed by staining with ethidium bromide, after
electrophoresis on 1% agarose gel and then purified using DNA fragment purification kit (Takara, Beijing, China). The purified DNA was recombined into plasmid pIRES2-EGFP using T4 ligase kit (Takara) and subjected
transformation into Ecoli DH5α according
to manufacture instruction. The successful construction was confirmed by PCR, EcoR I, and Sal I digestion and DNA sequencing.


#### 2.2.2. Preparations of Human Spermatozoa

Semen samples collected from the normal man were kept in aCO2 incubator (37°C/50 mL/L CO2 in air) for 30 minutes in order to be liquefied. Highly
motile spermatozoa were recovered from the semen with swim-up method. The sperm
suspension thereby obtained was centrifuged at 1500 rpm for 5 minutes. The
pellet was resuspended in BWW with 0.3% BSA and centrifuged again. The washed
spermatozoa were suspended in 5 mL of 10 μM ionophore solution for 10 minutes
to facilitate the capacitation and washed twice, and then suspended in BWW with
3.5% BSA to allow capacitation.


#### 2.2.3. Exposure of Spermatozoa to Plasmid pIRES2-EGFP-HBs

Two and half hours after the beginning of capacitation, the spermatozoa was exposed to plasmid pIRES2-EGFP-HBs. Briefly, a total of 100μL mixture
containing 1 μL plasmid pIRES2-EGFP-HBs (1.5 μg/ml), 6 μL liposome,
and 93 μL HEPES buffered saline was incubated at room temperature for 15 minutes, and then was added to sperm sample and kept in the incubator for another
1.5 hours. After that, the sperm sample was washed five times in 5 mL fresh
BWW.


#### 2.2.4. Preparation of Golden Hamster Oocytes

The mature hamsters were induced to superovulate by intraperitoneal
injection of 40 IU of  pregnant mare
serum gonadotropin (PMSG, Ningbo Hormone Product Co., Ltd., China) on day 1 of
oestrous cycle followed by administration of 40 IU  human chorionic gonadotrophin (hCG, Ningbo
Hormone product Co., Ltd.) 72 hours later. Superovulated oocytes were collected
from the ampullar region of oviducts 17 hours after hCG injection and freed
from cumulus cells in 0.1% hyaluronidase (Sigma). Cumulus-free oocytes
were washed twice in BWW andtreated with 0.1% trypsin (Sigma)to
remove the zona pellucida, then washed twice immediately in BWW.


#### 2.2.5. Insemination and Postinsemination Culture

Insemination was performed with the sperm suspensions at a concentration
of106/mL. The oocytes were kept in the sperm suspension for 20–30
minutes and then transferred and incubated in the sperm-free BWW under mineral
oil (Sigma) for another 1 hour to ensure sperm penetration. After washing
twice in OCM, the groups, containing five oocytes per group, were cultured in a
droplet (60 μL each) of OCM
under oil in a plastic Petri dish kept in a CO2 incubator (37°C, 50 mL/L CO2 in air) for 24 hours.


#### 2.2.6. Preparations of Embryos and Grouping

Twenty four hours after
insemination, two-cell embryos were collected and classified into two groups
under fluorescence microscope (Axiovert
40 CFL, Zeizz , Germany)
including group A with green fluorescence (Figure2(a)) and group B without green fluorescence (Figure 2(b)).Two-cell embryo with green fluorescence (a) and two-cell embryo without    green   fluorescence (b) 24 hours after insemination (arrows). 400x.
(a)(b)The two-cell embryos from group A and B were washed three times in cold
1X PBS to remove serum from the medium, and then each embryo was transferred singly
into a PCR tube for PCR and RT-PCR,
respectively.


#### 2.2.7. Single-Embryo PCR

Approximately, 4.5μL of cell lysis buffer were added to each
single-embryo of group A and B, mixed thenincubated at 70°C for 10 minutes. The cell lysate of embryo of group A and B was used as a DNA template, and pIRES2-EGFP-HBs and sterile ddH2O as the positive
and negative controls, respectively. All procedures of single-embryo PCR were the same as mentioned in Section 2.2.1.


#### 2.2.8. Single-Embryo RT-PCR

RNAwas extracted from each single-embryo of
groups A and B. Single-embryo RT-PCR was performed using the cells-cDNA II kit.
Briefly, 49.5μL ice-cold cell
lysis II buffer was added to each single-embryo sample and the mixture was incubated
at 75°C for 10 minutes. Approximately, 0.12 U DNase I
was added to the mixture which then incubated at 37°C for 30 minutes. To deactivate DNase, the samples
were incubated at 75°C for 5 minutes. Reverse transcription was preformed according to the kit
protocol. PCR amplification was
carried out with 5 μL cDNA
of each sample from reverse transcription reaction as a template, HBs specific
primer pair mentioned above, and two negative controls (minus template and
minus reverse transcription). The amplification step was carried out using the
same conditions of normal PCR to
amplify HBs gene as mentioned above. About 20 μL of each RT-PCR product were made visible by staining with EB after electrophoresis on 1.2%
agarose gel. This experiment was repeated three times under the same condition.


## 2.1. Materials

Biggers-Whitten-Whittingham (BWW) medium supplemented with 0.3% bovine
serum albumen (BSA, Sigma Chemicals Co., ST. Louis , MO) was
prepared for human sperm preparation, oocyte collection, insemination and
subsequent handling. Ovum culture medium (OCM, from Flow Laboratories, Germany)
containing 10% heat-inactivated fetal bovine serum was used in the post-insemination
culture.


## 2.2. Methods

### 2.2.1. Construction of Recombinant Plasmid pIRES2-EGFP-HBs

The expression vector for
HBs and EGFP was constructed as Figure1.Figure 1
Diagram of constructed recombinant plasmid pIRES2-EGFP-HBs.The HBs gene was amplified (403 bp) from plasmid pBR322-HBV by PCR and subcloned to plasmid pIRES2-EGFP to construct recombinant plasmid
pIRES2-EGFP-HBs. The PCR mixture consisted of 5μL 10X Taq buffer, 4 μL of
2.5 mM of MgCL2, 200 μmol/L of
each deoxynucleoside triphosphate (Invitrogen, Guangzhou, China), 20 pmol of each primer with EcoR I and
Sal I restriction sites (Takara Biotech, Japan) including forward: 5′-CGGAATTCTATCGCTGGATGTGTCTGC-3′ and reverse: 5′-GGTCGACAGACTTGGCCCCCAATAC-3′,
2.5 U Taq polymerase  and sterile ddH2O in
a final volume of 50 μL. The amplification program was as follows: denaturing
at 94°C for 5 minutes, and 35 cycles each at 94°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 45 seconds,
followed by a final extension at 72°C for 10 minutes. The amplification products were routinely analyzed by staining with ethidium bromide, after
electrophoresis on 1% agarose gel and then purified using DNA fragment purification kit (Takara, Beijing, China). The purified DNA was recombined into plasmid pIRES2-EGFP using T4 ligase kit (Takara) and subjected
transformation into Ecoli DH5α according
to manufacture instruction. The successful construction was confirmed by PCR, EcoR I, and Sal I digestion and DNA sequencing.


### 2.2.2. Preparations of Human Spermatozoa

Semen samples collected from the normal man were kept in aCO2 incubator (37°C/50 mL/L CO2 in air) for 30 minutes in order to be liquefied. Highly
motile spermatozoa were recovered from the semen with swim-up method. The sperm
suspension thereby obtained was centrifuged at 1500 rpm for 5 minutes. The
pellet was resuspended in BWW with 0.3% BSA and centrifuged again. The washed
spermatozoa were suspended in 5 mL of 10 μM ionophore solution for 10 minutes
to facilitate the capacitation and washed twice, and then suspended in BWW with
3.5% BSA to allow capacitation.


### 2.2.3. Exposure of Spermatozoa to Plasmid pIRES2-EGFP-HBs

Two and half hours after the beginning of capacitation, the spermatozoa was exposed to plasmid pIRES2-EGFP-HBs. Briefly, a total of 100μL mixture
containing 1 μL plasmid pIRES2-EGFP-HBs (1.5 μg/ml), 6 μL liposome,
and 93 μL HEPES buffered saline was incubated at room temperature for 15 minutes, and then was added to sperm sample and kept in the incubator for another
1.5 hours. After that, the sperm sample was washed five times in 5 mL fresh
BWW.


### 2.2.4. Preparation of Golden Hamster Oocytes

The mature hamsters were induced to superovulate by intraperitoneal
injection of 40 IU of  pregnant mare
serum gonadotropin (PMSG, Ningbo Hormone Product Co., Ltd., China) on day 1 of
oestrous cycle followed by administration of 40 IU  human chorionic gonadotrophin (hCG, Ningbo
Hormone product Co., Ltd.) 72 hours later. Superovulated oocytes were collected
from the ampullar region of oviducts 17 hours after hCG injection and freed
from cumulus cells in 0.1% hyaluronidase (Sigma). Cumulus-free oocytes
were washed twice in BWW andtreated with 0.1% trypsin (Sigma)to
remove the zona pellucida, then washed twice immediately in BWW.


### 2.2.5. Insemination and Postinsemination Culture

Insemination was performed with the sperm suspensions at a concentration
of106/mL. The oocytes were kept in the sperm suspension for 20–30
minutes and then transferred and incubated in the sperm-free BWW under mineral
oil (Sigma) for another 1 hour to ensure sperm penetration. After washing
twice in OCM, the groups, containing five oocytes per group, were cultured in a
droplet (60 μL each) of OCM
under oil in a plastic Petri dish kept in a CO2 incubator (37°C, 50 mL/L CO2 in air) for 24 hours.


### 2.2.6. Preparations of Embryos and Grouping

Twenty four hours after
insemination, two-cell embryos were collected and classified into two groups
under fluorescence microscope (Axiovert
40 CFL, Zeizz , Germany)
including group A with green fluorescence (Figure2(a)) and group B without green fluorescence (Figure 2(b)).Two-cell embryo with green fluorescence (a) and two-cell embryo without    green   fluorescence (b) 24 hours after insemination (arrows). 400x.
(a)(b)The two-cell embryos from group A and B were washed three times in cold
1X PBS to remove serum from the medium, and then each embryo was transferred singly
into a PCR tube for PCR and RT-PCR,
respectively.


### 2.2.7. Single-Embryo PCR

Approximately, 4.5μL of cell lysis buffer were added to each
single-embryo of group A and B, mixed thenincubated at 70°C for 10 minutes. The cell lysate of embryo of group A and B was used as a DNA template, and pIRES2-EGFP-HBs and sterile ddH2O as the positive
and negative controls, respectively. All procedures of single-embryo PCR were the same as mentioned in Section 2.2.1.


### 2.2.8. Single-Embryo RT-PCR

RNAwas extracted from each single-embryo of
groups A and B. Single-embryo RT-PCR was performed using the cells-cDNA II kit.
Briefly, 49.5μL ice-cold cell
lysis II buffer was added to each single-embryo sample and the mixture was incubated
at 75°C for 10 minutes. Approximately, 0.12 U DNase I
was added to the mixture which then incubated at 37°C for 30 minutes. To deactivate DNase, the samples
were incubated at 75°C for 5 minutes. Reverse transcription was preformed according to the kit
protocol. PCR amplification was
carried out with 5 μL cDNA
of each sample from reverse transcription reaction as a template, HBs specific
primer pair mentioned above, and two negative controls (minus template and
minus reverse transcription). The amplification step was carried out using the
same conditions of normal PCR to
amplify HBs gene as mentioned above. About 20 μL of each RT-PCR product were made visible by staining with EB after electrophoresis on 1.2%
agarose gel. This experiment was repeated three times under the same condition.


## 2.2.1. Construction of Recombinant Plasmid pIRES2-EGFP-HBs

The expression vector for
HBs and EGFP was constructed as Figure1.Figure 1
Diagram of constructed recombinant plasmid pIRES2-EGFP-HBs.The HBs gene was amplified (403 bp) from plasmid pBR322-HBV by PCR and subcloned to plasmid pIRES2-EGFP to construct recombinant plasmid
pIRES2-EGFP-HBs. The PCR mixture consisted of 5μL 10X Taq buffer, 4 μL of
2.5 mM of MgCL2, 200 μmol/L of
each deoxynucleoside triphosphate (Invitrogen, Guangzhou, China), 20 pmol of each primer with EcoR I and
Sal I restriction sites (Takara Biotech, Japan) including forward: 5′-CGGAATTCTATCGCTGGATGTGTCTGC-3′ and reverse: 5′-GGTCGACAGACTTGGCCCCCAATAC-3′,
2.5 U Taq polymerase  and sterile ddH2O in
a final volume of 50 μL. The amplification program was as follows: denaturing
at 94°C for 5 minutes, and 35 cycles each at 94°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 45 seconds,
followed by a final extension at 72°C for 10 minutes. The amplification products were routinely analyzed by staining with ethidium bromide, after
electrophoresis on 1% agarose gel and then purified using DNA fragment purification kit (Takara, Beijing, China). The purified DNA was recombined into plasmid pIRES2-EGFP using T4 ligase kit (Takara) and subjected
transformation into Ecoli DH5α according
to manufacture instruction. The successful construction was confirmed by PCR, EcoR I, and Sal I digestion and DNA sequencing.


## 2.2.2. Preparations of Human Spermatozoa

Semen samples collected from the normal man were kept in aCO2 incubator (37°C/50 mL/L CO2 in air) for 30 minutes in order to be liquefied. Highly
motile spermatozoa were recovered from the semen with swim-up method. The sperm
suspension thereby obtained was centrifuged at 1500 rpm for 5 minutes. The
pellet was resuspended in BWW with 0.3% BSA and centrifuged again. The washed
spermatozoa were suspended in 5 mL of 10 μM ionophore solution for 10 minutes
to facilitate the capacitation and washed twice, and then suspended in BWW with
3.5% BSA to allow capacitation.


## 2.2.3. Exposure of Spermatozoa to Plasmid pIRES2-EGFP-HBs

Two and half hours after the beginning of capacitation, the spermatozoa was exposed to plasmid pIRES2-EGFP-HBs. Briefly, a total of 100μL mixture
containing 1 μL plasmid pIRES2-EGFP-HBs (1.5 μg/ml), 6 μL liposome,
and 93 μL HEPES buffered saline was incubated at room temperature for 15 minutes, and then was added to sperm sample and kept in the incubator for another
1.5 hours. After that, the sperm sample was washed five times in 5 mL fresh
BWW.


## 2.2.4. Preparation of Golden Hamster Oocytes

The mature hamsters were induced to superovulate by intraperitoneal
injection of 40 IU of  pregnant mare
serum gonadotropin (PMSG, Ningbo Hormone Product Co., Ltd., China) on day 1 of
oestrous cycle followed by administration of 40 IU  human chorionic gonadotrophin (hCG, Ningbo
Hormone product Co., Ltd.) 72 hours later. Superovulated oocytes were collected
from the ampullar region of oviducts 17 hours after hCG injection and freed
from cumulus cells in 0.1% hyaluronidase (Sigma). Cumulus-free oocytes
were washed twice in BWW andtreated with 0.1% trypsin (Sigma)to
remove the zona pellucida, then washed twice immediately in BWW.


## 2.2.5. Insemination and Postinsemination Culture

Insemination was performed with the sperm suspensions at a concentration
of106/mL. The oocytes were kept in the sperm suspension for 20–30
minutes and then transferred and incubated in the sperm-free BWW under mineral
oil (Sigma) for another 1 hour to ensure sperm penetration. After washing
twice in OCM, the groups, containing five oocytes per group, were cultured in a
droplet (60 μL each) of OCM
under oil in a plastic Petri dish kept in a CO2 incubator (37°C, 50 mL/L CO2 in air) for 24 hours.


## 2.2.6. Preparations of Embryos and Grouping

Twenty four hours after
insemination, two-cell embryos were collected and classified into two groups
under fluorescence microscope (Axiovert
40 CFL, Zeizz , Germany)
including group A with green fluorescence (Figure2(a)) and group B without green fluorescence (Figure 2(b)).Two-cell embryo with green fluorescence (a) and two-cell embryo without    green   fluorescence (b) 24 hours after insemination (arrows). 400x.
(a)(b)The two-cell embryos from group A and B were washed three times in cold
1X PBS to remove serum from the medium, and then each embryo was transferred singly
into a PCR tube for PCR and RT-PCR,
respectively.


## 2.2.7. Single-Embryo PCR

Approximately, 4.5μL of cell lysis buffer were added to each
single-embryo of group A and B, mixed thenincubated at 70°C for 10 minutes. The cell lysate of embryo of group A and B was used as a DNA template, and pIRES2-EGFP-HBs and sterile ddH2O as the positive
and negative controls, respectively. All procedures of single-embryo PCR were the same as mentioned in Section 2.2.1.


## 2.2.8. Single-Embryo RT-PCR

RNAwas extracted from each single-embryo of
groups A and B. Single-embryo RT-PCR was performed using the cells-cDNA II kit.
Briefly, 49.5μL ice-cold cell
lysis II buffer was added to each single-embryo sample and the mixture was incubated
at 75°C for 10 minutes. Approximately, 0.12 U DNase I
was added to the mixture which then incubated at 37°C for 30 minutes. To deactivate DNase, the samples
were incubated at 75°C for 5 minutes. Reverse transcription was preformed according to the kit
protocol. PCR amplification was
carried out with 5 μL cDNA
of each sample from reverse transcription reaction as a template, HBs specific
primer pair mentioned above, and two negative controls (minus template and
minus reverse transcription). The amplification step was carried out using the
same conditions of normal PCR to
amplify HBs gene as mentioned above. About 20 μL of each RT-PCR product were made visible by staining with EB after electrophoresis on 1.2%
agarose gel. This experiment was repeated three times under the same condition.


## 3. Results

### 3.1. Construction of Recombinant Plasmid pIRES2-EGFP-HBs

Identification of recombinant plasmid pIRES2-EGFP-HBs was shown as Figure3. The successful construction was confirmed by PCR, EcoR I, and Sal I digestion, DNA sequencing, and BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST).Identification of the recombinant plasmid pIRES2-EGFP-HBs. (a) PCR result of
HBs gene before transformation. M: Markers; 1: HBs gene; 2: negative control. (b) PCR
result of HBs gene after transformation. M: Markers; 1 and 2: HBs gene; 3: negative control
after transformation. (c) Result of enzyme digestion. M: Markers; 1 and 2: pRIS2-EGFP-HBs
digested using EcoiR I and Sal I after transformation; 3: pRIS2-EGFP empty as native
control.
(a)(b)(c)


### 3.2. The Rate of Two-Cell Embryos

A total of 350 zona-free golden hamster oocytes were fertilized with human
spermatozoa in this study. 201 embryos were obtained including 105 one-cell and
96 two-cell embryos (Figures4(a) and 4(b)). The fertilization rate was 57.42% (201/350). About 47.76% (96/201)
fertilized eggs could develop into two-cell stage of embryonic development. 33
of them (34.37%) showed green fluorescence.One-cell embryo showing a male and a female pronuclei (a), and two-cell embryo
one nucleus in each, (b) 24 hours after insemination (arrows). 400 X, under phase contrast
microscope.
(a)(b)


### 3.3. Single-Embryo PCR Results

Single-embryo PCR results are shown in Figure5. The HBs DNA positive bands (403 bp) were detected in three
samples from single embryos with green fluorescence and the positive controls, and
not detected in three samples from single embryos without green fluorescence
and the negative control. All amplification products were found to be
reproducible when reactions repeated three times using the same reaction
conditions.Figure 5
PCR product of HBs gene: M: Markers; 1, 2, and 3: the samples from three single
embryos with green fluorescence showing HBs DNA positive bands, respectively; 4, 5, and 6:
the samples from three single embryos without green fluorescence showing negative results
for HBs DNA; 7: the positive control; 8: the negative control.


### 3.4. Single-Embryo RT-PCR Results

Single-embryo RT-PCR results are shown in Figure6. The HBs cDNA
positive bands (403 bp) were detected in three samples from single embryos with
green fluorescence, and not
detected in three samples from single embryos without green fluorescence and
the negative controls (−RT and −T). All
amplification products were found to be reproducible when reactions repeated
three times using the same reaction conditions.Figure 6
RT-PCR product of HBs gene: M: Markers; 1, 2, and 3: the samples from three
single embryos with green fluorescence showing HBs cDNA positive bands, respectively; 4,
5, and 6: the samples from three single embryos without green fluorescence showing negative
results for HBs DNA; 7: the negative control (−RT); 8: the negative control (−T).


## 3.1. Construction of Recombinant Plasmid pIRES2-EGFP-HBs

Identification of recombinant plasmid pIRES2-EGFP-HBs was shown as Figure3. The successful construction was confirmed by PCR, EcoR I, and Sal I digestion, DNA sequencing, and BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST).Identification of the recombinant plasmid pIRES2-EGFP-HBs. (a) PCR result of
HBs gene before transformation. M: Markers; 1: HBs gene; 2: negative control. (b) PCR
result of HBs gene after transformation. M: Markers; 1 and 2: HBs gene; 3: negative control
after transformation. (c) Result of enzyme digestion. M: Markers; 1 and 2: pRIS2-EGFP-HBs
digested using EcoiR I and Sal I after transformation; 3: pRIS2-EGFP empty as native
control.
(a)(b)(c)


## 3.2. The Rate of Two-Cell Embryos

A total of 350 zona-free golden hamster oocytes were fertilized with human
spermatozoa in this study. 201 embryos were obtained including 105 one-cell and
96 two-cell embryos (Figures4(a) and 4(b)). The fertilization rate was 57.42% (201/350). About 47.76% (96/201)
fertilized eggs could develop into two-cell stage of embryonic development. 33
of them (34.37%) showed green fluorescence.One-cell embryo showing a male and a female pronuclei (a), and two-cell embryo
one nucleus in each, (b) 24 hours after insemination (arrows). 400 X, under phase contrast
microscope.
(a)(b)


## 3.3. Single-Embryo PCR Results

Single-embryo PCR results are shown in Figure5. The HBs DNA positive bands (403 bp) were detected in three
samples from single embryos with green fluorescence and the positive controls, and
not detected in three samples from single embryos without green fluorescence
and the negative control. All amplification products were found to be
reproducible when reactions repeated three times using the same reaction
conditions.Figure 5
PCR product of HBs gene: M: Markers; 1, 2, and 3: the samples from three single
embryos with green fluorescence showing HBs DNA positive bands, respectively; 4, 5, and 6:
the samples from three single embryos without green fluorescence showing negative results
for HBs DNA; 7: the positive control; 8: the negative control.


## 3.4. Single-Embryo RT-PCR Results

Single-embryo RT-PCR results are shown in Figure6. The HBs cDNA
positive bands (403 bp) were detected in three samples from single embryos with
green fluorescence, and not
detected in three samples from single embryos without green fluorescence and
the negative controls (−RT and −T). All
amplification products were found to be reproducible when reactions repeated
three times using the same reaction conditions.Figure 6
RT-PCR product of HBs gene: M: Markers; 1, 2, and 3: the samples from three
single embryos with green fluorescence showing HBs cDNA positive bands, respectively; 4,
5, and 6: the samples from three single embryos without green fluorescence showing negative
results for HBs DNA; 7: the negative control (−RT); 8: the negative control (−T).


## 4. Discussion

In the present study, the sensitive and rapid assay was
established to explore the feasibility of virus vertical transmission via male germ
line based on the work of many scientists. It consisted of the following
procedures: the construction of recombinant
plasmid pIRES2-EGFP-HBs, the exposure of human sperm to
virus DNA, interspecific in vitro fertilization between human
sperm and zona-free golden hamster ova, identifiable embryos with
sperm-mediated virus genes, single-embryo PCR, and single-embryo RT-PCR.We successfully constructed the recombinant
plasmid pIRES2-EGFP-HBs (Figure1), which was confirmed by
PCR, EcoR I and Sal I digestion, DNA sequencing, and BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST) (Figure 3). This plasmid contains HBs and EGFP genes, which expressed EGFP as
reporter for HBs gene expression. It enables us to distinguish between the embryos
with and without HBs gene through observation of the presence of green
fluorescence (Figure 2), and to demonstrate the existence and expression of
human sperm-mediated HBs genes in host embryonic cells (Figures 5 and 6).
Interspecific in vitro
fertilization between human sperm and zona-free golden hamster ova established
by Yanagimachi et al. [20] was widely used in the research on reproductive
biology. We extended its application to the area of virus transmitting research
and introduced it into the present assay.In theory, the existence and expression of HBs gene should be detected
in two-cell embryos with green fluorescence and not detected in those without
green fluorescence. The results of single-embryo PCR and single-embryo RT-PCR confirmed
our assumption. HBs DNA positive
bands were detected in the embryos with green fluorescence (PCR and RT-PCR)
and positive control (PCR) and not
observed in the embryos without green fluorescence and the negative controls (PCR and RT-PCR)
(Figures5 and 6). The
same results were obtained in the repeated experiments which demonstrated the exactness
and efficiency of our assay as well as the reliability of
our previous research results, that is, the sperm-mediated HBV genes are able
to express their functions in host early embryonic cells [16–19, 21].Zona
pellucida is a glycoprotein coat surrounding the egg proper. It protects the
fragile eggs and embryos from physical damage [22].
We use zona-free oocytes in the current
experimental assay because our previous studies have confirmed that the zona
pellucida could not become a barrier to the entry of virus DNA sequences into the oocytes and virus DNA fragments were able to enter into oocytes and
integrate into their chromosomes either in vitro or in vivo experiments [16–19].The
present assay has the following advantages. (1) The researchers can exactly
know which embryo contains human sperm-mediated virus genes through
observation of green fluorescence and avoid the blindness occurred in the previous
experiments. The results which were obtained by performing many experiments in
the previous studies could be gained just in one experiment now. Thus, it saves
much time and energy.  (2) The test
sample needed a batch of embryos in the previous studies and now a single
embryo already meets the needs of experiments in the present study. It not only
economized the experimental materials but also made experimental results more
exact and reliable. (3) The present assay would make it easy to explore vertical
transmission of other viruses (X) via human spermatozoa if recombinant plasmid pIRES2-EGFP-HBs was replaced by pIRES2-EGFP-X. (4)
The present assay also avoided the moral, ethical, and methodological problems
which would present when the vertical transmission of sperm-mediated
virus genes were studied using embryos from fertilization between human oocytes and spermatozoa.With
progresses of virology, epidemiology, molecular biology, and genetics, more and
more viruses have been found to be associated with human health. The studies on the transmission of viruses are of
substantial importance in virology as well as in public health. Our study provided a very useful assay for
study on the exact vertical transmission of viruses via the germ line. It might
open a wide area for basic and clinical research because severe
threats to humanity are posed by many viruses, such as HCV, HIV, SARS, and the like.



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*Source: 495436-2008-07-22.xml*