quantitative analysis of porcine endogenous retroviruses in different organs of transgenic pigs...

Quantitative Analysis of Porcine Endogenous Retroviruses

in Different Organs of Transgenic Pigs Generated

for Xenotransplantation

Urszula Mazurek • Magdalena C. Kimsa • Barbara Strzalka-Mrozik •

Malgorzata W. Kimsa • Jolanta Adamska • Daniel Lipinski • Joanna Zeyland •

Marlena Szalata • Ryszard Slomski • Jacek Jura • Zdzislaw Smorag •

Roman Nowak • Joanna Gola

Received: 1 February 2013 / Accepted: 29 April 2013

� Springer Science+Business Media New York 2013

Abstract The pig appears to be the most promising ani-

mal donor of organs for use in human recipients. Among

several types of pathogens found in pigs, one of the greatest

problems is presented by porcine endogenous retroviruses

(PERVs). Screening of the source pig herd for PERVs

should include analysis of both PERV DNA and RNA.

Therefore, the present study focuses on quantitative anal-

ysis of PERVs in different organs such as the skin, heart,

muscle, and liver and blood of transgenic pigs generated

for xenotransplantation. Transgenic pigs were developed to

express the human a-galactosidase, the human a-1,2-fu-

cosyltransferase gene, or both genetic modifications of the

genome (Lipinski et al., Medycyna Wet 66:316–322, 2010;

Lipinski et al., Ann Anim Sci 12:349–356, 2012; Wiecz-

orek et al., Medycyna Wet 67:462–466, 2011). The copy

numbers of PERV DNA and RNA were evaluated using

real-time Q-PCR and QRT-PCR, respectively. Compara-

tive analysis of all PERV subtypes revealed the following

relationships: PERV A[ PERV B[PERV C. PERV A

and B were found in all samples, whereas PERV C was

detected in 47 % of the tested animals. The lowest level of

PERV DNA was shown in the muscles for PERV A and B

and in blood samples for PERV C. The lowest level of

PERV A RNA was found in the skin, whereas those of

PERV B and C RNA were found in liver specimens.

Quantitative analysis revealed differences in the copy

number of PERV subtypes between various organs of

transgenic pigs generated for xenotransplantation. Our data

support the idea that careful pig selection for organ dona-

tion with low PERV copy number may limit the risk of

retrovirus transmission to the human recipients.

Introduction

Xenotransplantation includes any procedure that involves

the transplantation, implantation, or infusion into a human

recipient of live cells, tissues, or organs from a non-human

animal source or human body fluids, cells, tissues, or

organs that have had ex vivo contact with live non-human

animal cells, tissues, or organ [10]. The pig appears to be

the most promising animal donor of organs for use in

human recipients [5]. However, various immunological and

non-immunological barriers still need to be overcome.

The possibility of animal pathogen transmission with

transplanted organs is a safety issue to be considered when

using pigs for xenotransplantation. Among several types of

pathogens found in pigs, one of the greatest problems is

presented by porcine endogenous retroviruses (PERVs)

[26]. Three replication-competent classes of PERVs

(PERV A, PERV B, and PERV C) have been identified in

U. Mazurek � M. C. Kimsa (&) � B. Strzalka-Mrozik �

M. W. Kimsa � J. Adamska � J. Gola

Department of Molecular Biology, Medical University of Silesia,

Narcyzow 1, Sosnowiec, Poland

e-mail: [email protected]

D. Lipinski � J. Zeyland � M. Szalata � R. Slomski

Department of Biochemistry and Biotechnology, Poznan

University of Life Sciences, Wolynska 35, Poznan, Poland

D. Lipinski � M. Szalata � R. Slomski

Institute of Human Genetics, Polish Academy of Sciences,

Strzeszynska 32, Poznan, Poland

J. Jura � Z. Smorag

Department of Animal Reproduction, The National Research

Institute of Animal Production, Krakowska 1, Balice, Poland

R. Nowak

Department and Clinic of Orthopaedics, Medical University of

Silesia, Sosnowiec, Poland

123

Curr Microbiol

DOI 10.1007/s00284-013-0397-3

the genomic DNA of pigs [30]. PERVs use RNA as their

genetic material, but DNA is used in the virus replication

cycle, and this can be integrated into the host DNA as a

provirus [20]. Therefore, screening of the source pig herd

for PERVs should include analysis of both DNA and RNA.

The most profound barrier when using pig organs is the

host immunological rejections [11]. Transgenic pigs, such

as pigs expressing the human membrane-bound comple-

mentary proteins, pigs expressing the human a-1,2-fuco-

syltransferase, the human a-galactosidase (aGal), and

alpha-1,3-galactosyltransferase knockout pigs, have been

generated to achieve resistance against xenograft rejection

[9]. However, there is no information as to how integration

of transgene in the pig genomes may change the infectivity

and replication activity of PERVs.

The International Xenotransplantation Association

specified the conditions which must be taken in clinical

trials with pig cells. They are as follows: careful screening

of the source pig herd for PERVs, selection of pigs which

exhibit low level expression of PERV A and PERV B,

selection of pigs which do not contain PERV C in their

germ line, and screening of xenotransplant recipients for

PERV transmission [7].

In light these observations and recommendations, our

study focused on quantitative analysis of PERVs in different

organs of transgenic pigs generated for xenotransplantation.

Materials and Methods

Transgenic Pig Production

Porcine peripheral blood samples and tissue fragments

were obtained from 3- to 6-month-old transgenic Polish

Landrace pigs originating from an experimental farm of the

National Research Institute of Animal Production (Zerniki

Wielkie, Poland) (Table 1).

Transgenic pigs were produced by pronuclear DNA

microinjection and were developed to express the human

a-galactosidase, the human a-1,2-fucosyltransferase gene

(FUT II), or both genetic modifications of the genome

(FUT II 9 aGal) [17, 18, 31]. The characterization of

transgenic animals was performed on four levels including

screening for the presence of transgene using PCR, deter-

mination of transgene expression by RT-PCR, mapping of

transgene by fluorescent in situ hybridization (FISH), and

measurement of transgene activity by flow cytometry as

described previously by Lipinski et al. [17, 18] and Wie-

czorek et al. [31].

Preparation of Porcine Tissues

To quantitative analyze the PERVs, 33 fragments of the

skin, heart, muscle, and liver and 71 blood samples of

transgenic pigs were used. Porcine tissues were rinsed with

buffered sterile physiological saline solution (pH 6.5) to

remove residue blood and subsequently transported to the

laboratory in buffered sterile physiological saline solution

(pH 6.5) at 4 �C. Blood samples were collected on EDTA

solution. All samples were stored frozen at -80 �C for

48 h until DNA and RNA extraction. The study was

approved by the ethical committee of the Polish State

Committee of Scientific Research.

DNA and RNA Extraction

Genomic DNA was isolated from tissues fragments using a

salting out extraction method [6] and from blood using a

commercially available kit, QIAamp DNAMini Kit (Qiagen,

Valencia, CA), according to the manufacturer’s instructions.

Total RNAwas extracted from tissues using a TRIzol reagent

(Invitrogen, Carlsbad, CA) and from blood using the phenol–

chloroform method [3]. In order to exclude possible RNA

contamination, DNA extracts were treated with RNase A

(MBI Fermentas, Vilnius, Lithuania). RNA extracts were

treated with DNase I (RNeasy Mini Kit, Qiagen, Valencia,

CA), according to themanufacturer’s instructions. The quality

of extracts was checked electrophoretically using 0.9 %

agarose gel stained with ethidium bromide (Sigma-Aldrich,

St. Louis,MO). The results were analyzed and recorded using

the 1D Bas-Sys gel documentation system (Biotech-Fisher,

Perth, Australia). Nucleic acid concentration was determined

using a GeneQuant II RNA/DNA spectrophotometer (Phar-

macia Biotech, Cambridge, UK).

Quantitative PCR and RT-PCR Assay

Detection of the copy number of PERVs was performed as

described previously [6, 16]. The copy numbers of PERV

Table 1 Selected features of the screened transgenic pigs

Characteristics Animals (n = 71)

Transgene

aGal 27

FUT II 22

FUT II 9 aGal 22

Gender

Female 35

Male 36

Age (months) 4.5 (3–6)

Weight (kg) 49.5 (25–87)

Values of parameters are expressed as means (minimum–maximum)

U. Mazurek et al.: Quantitative Analysis of PERVs

123

A, PERV B, and PERV C DNA and PERV A, PERV B,

and PERV C RNA were evaluated using real-time Q-PCR

and SYBR Green I chemistry (SYBR Green QuantiTect

PCR Kit, QIAGEN, Valencia, CA) and real-time QRT-

PCR and SYBR Green I chemistry (SYBR Green Quanti-

Tect RT-PCR Kit, Qiagen, Valencia, CA), respectively.

Porcine cellular GAPDH gene (glyceraldehyde-3-phos-

phate dehydrogenase) was included as an endogenous

positive control of the amplification and integrity of

extracts. Normal porcine kidney epithelial cells (PK15 cell

line) were used also as a positive control for PERV A and

PERV B sequences. The analyses were performed using an

OpticonTM DNA Engine Continuous Fluorescence Detec-

tor (MJ Research, Watertown, MA). All samples were

tested in triplicate. Oligonucleotide primers specific for

PERV A, PERV B, PERV C, and GAPDH were described

previously by Bosch et al. [2], Machnik et al. [22], and

Moon et al. [24] (Table 2).

The thermal profile for Q-PCR included polymerase

activation at 95 �C for 15 min and then 40 cycles con-

sisting of the following temperatures and time intervals:

94 �C for 30 s, 65 �C for 45 s, and 72 �C for 40 s. The

thermal profile for one-step QRT-PCR included reverse

transcription at 50 �C for 30 min, polymerase activation at

95 �C for 15 min, and then 30 cycles consisting of the

following temperatures and time intervals: 94 �C for 30 s,

65 �C for 45 s, and 72 �C for 40 s.

The point at which a PCR product is first detected above

a fixed threshold, termed a cycle threshold (Ct), was

determined for each sample, and an average Ct of triplicate

samples was calculated. To quantify the results obtained by

PCR and RT-PCR, a standard curve method was used,

described previously by Cyganek-Niemiec et al. [6] and

Strzalka-Mrozik et al. [28], respectively. Specificity of the

PCR reaction was confirmed by determining the charac-

teristic melting temperature (Tm) of each amplimer, and

the PCR products were separated on 6 % polyacrylamide

gels (PAA) and visualized with silver salts.

The obtained results of PERV DNA copy number were

shown as copies per cell. For all calculations, it was

assumed that a pig cell contained the same amount of

genomic DNA as a human cell (6 pg DNA/cell). The

obtained results of the RNA copy number of PERVs were

recalculated per 1 lg of total RNA.

Statistical Analyses

Statistical analyses were performed using Statistica 9.0

software (StatSoft, Tulsa, OK), and the level of signifi-

cance was set at P\ 0.05. Values were expressed as

median (Me) with the 25th and 75th quartiles. For statis-

tical analyses of PERV copy number, non-parametric tests

were used because the Shapiro–Wilk test indicated that the

data were not normally distributed. The Kruskal–Wallis

test, post hoc multiple test based on average ranks, and the

Mann–Whitney U test were applied to compare the copy

number of PERV DNA and RNA in porcine tissues.

Results

Characterization of Transgenic Animals

The presence of transgenes and the expression of human

a-1,2-fucosyltransferase and human aGal genes in tissues

of pigs were confirmed by PCR and RT-PCR (Figs. 1, 2 for

aGal data not shown). Moreover, flow cytometry revealed

that in transgenic pigs expressing human a-1,2-fucosyl-

transferase, four times decrease of Gal antigen expression

(40.68) in comparison with non-transgenic pigs (171.54)

was observed.

Table 2 Characteristics of

primers used for real-time

Q-PCR and QRT-PCR

bp Base pairs, Tm melting

temperature, F forward,

R reverse, I inosine bases

Gene Sequence of primers Length of

amplicon (bp)

Tm

(�C)

PERV env A F: 50-GAGATGGAAAGATTGGCAACAGCG-30

R: 50-AGTGATGTTAGGCTCAGTGGGGAC-30

364 80.0

PERV env B F: 50-AATTCTCCTTTGTCAATTCCGGCCC-3

R: 50-CCAGTACTTTATCGGGTCCCACTG-30

270 81.0

PERV env C F: 50-CTGACCTGGATTAGAACTGGAAGC-30

R: 50-GTTATGTTAGAGGATGGTCCTGGTC-30

284 80.4

PERV env C F: 50-TCTATACGTTTGCCTCAGATCAGTIIIIIC

TAGTCTG-30

R: 50-CCAGGTCAGGTAATTAAATTGTCCIIIII

TGGTATAGG-30

262 79.0

pGAPDH F: 50-TGTCGCCATCAATGACCCC-30

R: 50-TGACAAGCTTCCCATTCTC-30

295 80.1


123

Specificity of the Quantitative PCR and RT-PCR Assay

Q-PCR and QRT-PCR specificity for the target genes was

confirmed experimentally on the basis of amplimers’

melting temperatures. For each PCR product, a single peak

at expected temperatures was observed: PERV A at

80.0 �C, PERV B at 81.0 �C, PERV C at 80.4 �C, and

GAPDH at 80.1 �C. (data not shown). Gel electrophoresis

also revealed the presence of a single product of predicted

length (data not shown).

Detection of PERVs in Studied Herd of Pigs

The genotyping of PERVs was performed by Q-PCR and

QRT-PCR using specific primers for envA, envB, and envC

genes. PERV A and B proviral DNA was found in all

samples of pig genomic DNA, whereas PERV C DNA was

detected in 47 % of the tested animals. Similarly, the

expression of PERV A and PERV B at the RNA level was

positively detected in all tested samples, while PERV C

subtype was detected in 47 % of pigs.

Quantitative relations between the DNA and RNA copy

number of the different subtypes of PERVs in screened herd

of pigs were evaluated. Comparative analysis of all PERV

subtypes in terms of PERV DNA copy number/cell revealed

the following relationships in all tested tissues (blood, skin,

heart, muscle, and liver): PERV A[ PERV B (P\ 0.001,

post hoc test); PERV A[ PERV C (P\ 0.001, post hoc

test); PERV B[ PERV C (P\ 0.001, post hoc test)

(Table 3). The quantitative relations between RNA of these

three PERV subtypes were similar: PERV A[ PERV B

(P\ 0.001, post hoc test); PERVA[ PERV C (P\ 0.001,

post hoc test); PERVB[ PERVC (P\ 0.001, post hoc test)

(Table 3).

Distribution of PERVs in Different Porcine Organs

Different tissue fragments from the skin, heart, muscle,

liver, and blood of transgenic animals were analyzed to

determine organ-specific differences in PERV copy

number and PERV expression. Among all analyzed tissues,

the highest level of PERV A DNA was observed in blood

samples (Me = 59.3 copies/cell). In the case of PERV B

and PERV C DNA, the highest level was demonstrated in

heart samples (Me = 19.8 copies/cell, Me = 1.3 copies/

cell, respectively). The lowest level of PERV DNA was

shown in the muscles for PERV A and PERV B

(Me = 21.7 copies/cell, Me = 13.0 copies/cell, respec-

tively) and in blood samples for PERV C (Me = 0.005

copies/cell). Differences between investigated tissues were

statistically significant for PERV A (P\ 0.0037, Kruskal–

Wallis test) and PERV C (P\ 0.0031, Kruskal–Wallis

test). There was no statistically significant differences for

PERV B (P = 0.054, Kruskal–Wallis test) (Fig. 3a, b, c).

A comparable analysis of the PERVRNA copy number in

porcine tissues revealed that the highest level of three sub-

types of PERVs was in blood samples (Me = 1,473,249.8

copies/lg RNA for PERV A, Me = 471,517.9 copies/lg

RNA for PERV B, Me = 19,918.9 copies/lg RNA for

PERV C). The lowest level of PERV A RNA was found in

skin samples (Me = 31,372.7 copies/lg RNA). In the case

of PERV B and PERV C, the lowest RNA copy number in

liver specimens was noted (Me = 40,960.1 copies/lg RNA,

Me = 3,079.1 copies/lg RNA, respectively). Significant

differenceswere found for PERVAand PERVBRNA levels

between tested tissues (P\ 0.001, P\ 0.001, Kruskal–

Wallis test, respectively). There were no statistically sig-

nificant differences for PERV C (P = 0.824, Kruskal–

Wallis test) (Fig. 4a, b, c).

Distribution of PERVs Among Individuals of Screened

Herd

In the next step of the study, the presence of PERV proviral

DNA and PERV expression was evaluated between

Fig. 1 Analysis of CMVFUT transgene integration; lane M size

marker (kDNA/EcoRI, HindIII); lane 0 negative control 1 (no DNA);

lane ? positive control (pCMVFUT gene construct); lane NT

negative control (DNA of non-transgenic pig). Analysis results:

animals numbered 754, 756, 759, and 761 have integrated transgene

(positive result); remaining animals were non-transgenic

Fig. 2 Analysis of expression of human a-1,2-fucosyltransferase

gene in tissues of transgenic female. The arrow indicates PCR

products showing expression of human a-1,2-fucosyltransferase gene

under control of the CMV promoter. Lanes 1–5: transgenic pig; lanes

6–10: non-transgenic pig. Lanes 1 and 6 heart; lanes 2 and 7 kidney;

lanes 3 and 8 liver; lanes 4 and 9 muscle; lanes 5 and 10 ovary; lane

11 negative control (no DNA); lane 12 positive control (pCMVFUT

gene construct); lane 13 size marker (k DNA/HindIII, EcoRI)


123

individuals of the screened herd for all tested tissues. The

copy number of PERVA, PERVB, and PERV C in genomic

DNA varied significantly in different tissues among indi-

vidual Polish Landrace pigs (P\ 0.001, Kruskal–Wallis test

for three PERV subtypes) (Fig. 5). The PERV A, PERV B,

and PERV C expression at RNA level also varied signifi-

cantly in different individuals (P\ 0.001, Kruskal–Wallis

test for three PERV subtypes) (Fig. 6). Additionally, there

were no observed significant differences in GAPDH level in

the examined samples (Fig. 7).

Differences in the Copy Number of PERVs Between

Males and Females

The DNA and RNA copy number of particular PERV

subtypes depending on the gender of pigs was analyzed.

There was no statistically significant difference between

the copy number of PERV A, PERV B, PERV C DNA and

RNA in tested males and females (P[ 0.05, Mann–

Whitney U test).

Differences in the Copy Number of PERVs

in Transgenic Pigs

During the next stage of the study, differences in the copy

number of PERV subtypes between transgenic pigs were

evaluated. There was no significantly different copy num-

ber of PERV A, PERV B, PERV C DNA and expressions

of PERV A, PERV B, PERV C among pigs with the human

aGal, pigs expressing the human a-1,2-fucosyltransferase

gene (FUT II), or pigs with both genetic modifications of

the genome (FUT II 9 aGal) (P[ 0.05, Kruskal–Wallis

test).

Table 3 The quantitative relations between the copy number of subtypes of PERV DNA and PERV RNA in tested animals

PERV A PERV B PERV C P*

PERV DNA/cell 48.3 (21.6–109.5) [ 17.6 (2.3–43.0) [ 0.04 (0.003–2.7) P\ 0.0001

PERV RNA/lg

RNA

441,735.8 (70,948.6–1,894,397.5) [ 203,815.9 (22,125.5–906,351.0) [ 6,431.9 (1,232.1–53,743.3) P\ 0.0001

Statistical significance: *P\ 0.05, Kruskal–Wallis test Median with the 25th and 75th quartiles are presented

Fig. 3 Comparison of the copy number of PERV A (a), PERV B (b), and PERV C (c) DNA between different porcine tissues. Box and whisker

plots present medians ± quartiles and extreme values of copy numbers per cell; *P\ 0.05 versus blood, post hoc test

Fig. 4 Comparison of the copy number of PERV A (a), PERV B (b), and PERV C (c) RNA between different porcine tissues. Box and whisker

plots present medians ± quartiles and extreme values of copy numbers per 1 lg of total RNA; *P\ 0.05 versus blood, post hoc test


123

Discussion

The transplantation of porcine organs, tissues, or cells is

associated with the potential risk of various pathogen

infections. PERVs, permanently integrated into the genome

of pigs, may be extremely important in this respect. PERV

A and PERV B are polytropic viruses and they can infect

human cells in vitro [30, 32]. PERV C can infect only

porcine cells and is not present in the genomes of all pigs

[30]. PERV infection and their propagation in human

Fig. 5 Comparison of the copy number of PERV A, PERV B, and PERV C DNA between individuals of screened herd in different porcine

tissues


123

recipients cannot be ruled out after xenotransplantation.

Therefore, the selection of pigs with possible low levels of

PERV expression is required.

Our results revealed that PERV DNA is constantly

present in the genome of pigs regardless of tissue type,

which is in agreement with other published results when

the examined material constituted porcine tissues, but from

non-transgenic pig [13, 29]. In the present study, the real-

time Q-PCR and QRT-PCR techniques were used to

evaluate the copy number of PERV A, PERV B, and PERV

C DNA and RNA in porcine tissues. Our results showed

that PERV A and PERV B DNA and RNA were present in

Fig. 6 Comparison of the copy number of PERV A, PERV B, and PERV C RNA between individuals of screened herd in different porcine

tissues


123

whole tested herd. The other authors also demonstrated the

presence of PERVs, e.g., in aortic, pulmonary heart valves

and cardiac muscles obtained from pigs [25]. However, no

quantitative correlation between subtypes of PERVs was

found [25]. In the next step of our research, we indicated

that copy number of PERV subtypes was different between

analyzed tissues. Sypniewski et al. [29] also studied the

distribution of PERVs in different pig organs and showed

that the kidney may be potentially the biggest PERV res-

ervoir. In turn, in our study, the highest level of PERV

DNA was revealed in blood and heart samples. It seems

that some lower-PERV copy tissues have less opportunity

Fig. 7 The GAPDH level in different porcine tissues among individuals of screened herd


123

to express infectious PERVs than those that carry more

PERV copies [33]. Furthermore, Bittmann et al. [1] found

expression of the viral mRNA and proteins in many pig

organs at different levels. In contrast to our results, a high

PERV expression was indicated in lymphoid organs, while

in the other organs such as the pancreas, liver, and in the

ovarian tissue, the expression was low. The results we have

obtained revealed a lower copy number of PERV RNA in

organs such as the skin, heart, muscle, and liver compared

to blood samples. Moreover, Semaan et al. [27] detected

that PERV expression was the highest in the spleen and

lungs and the lowest in the liver and heart. These findings

may suggest that selection of donor animals for future

clinical xenotransplantation should be organ specific.

Interestingly, Liu et al. [19] indicated that PERV copy

number in genomic DNA and PERV expression at the

RNA level varied significantly among individuals of the

same breed, which is in agreement with our findings. The

reasons for these observations may be genetic or age-

related differences and differences in environmental

exposures [33]. For some individuals, the PERV copy

number in genomic DNA and the PERV expression at the

RNA level were low. Likewise, Dieckhoff et al. [8]

observed very low expression of PERVs in tested pigs.

Additionally, in our research, for some pigs, PERV DNA

copy number was higher than PERV expression at the RNA

level. It can be explained that only intact PERV DNA

copies can be transcripted into functional PERV retrovirus

RNA [23]. On the other hand, for some pigs, we observed a

higher PERV RNA than PERV DNA copy numbers. It can

suggest that although the PERV DNA copy number per cell

was low, porcine cells may be able to produce more new

infectious retroviral particles. Likewise, Liu et al. [19]

observed imparity between PERV DNA and RNA copy

numbers for some individuals of Chinese experimental

miniature pigs. These data imply that it is possible to select

animals with low PERV load, which is beneficial for the

safety of xenotransplantation.

The choice of PERV C-free animals is advantageous to

transplantation safety in view of the reduced risk of the

emergence of PERV A/C recombinants, which exhibit

increased infectivity [12]. In previous studies, 97.2 %

animals carried PERV C in the germ line and 28.1 %

animals carried PERV A/C in the genome of lymphoid

cells, but not in the germ line [8]. In the studies performed

by Kaulitz et al. [15], 92 % of the German landrace and

transgenic pigs were found to be positive for PERV C. As

shown in the previously published data [8, 14, 15], the

number of PERV C-negative animals was low. Impor-

tantly, in our research, PERV C occurred only in 47 % of

samples. Similarly, Liu et al. [19] showed the presence of

the PERV C subtype in 30 % of analyzed pigs. Thus, the

PERV infection risk can be reduced by screening individ-

uals not containing PERV C.

In order to evaluate the prevalence of PERV C and to

identify PERV C-free animals, different methods were

developed [15]. In the present study, to assess the copy

number of PERV C DNA and RNA, primers complemen-

tary to envC gene and primers with inosine bases were used

[24]. The latter oligonucleotides are constructed from two

segments joined by a polydeoxyinosine linker. The 50

segment is 18–25 bases in length and preferentially binds

to the template DNA, initiating stable annealing. The 30

segment is 6–12 bases in length and selectively binds to its

target, blocking non-specific annealing. The poly(I) linker

blocks extension of primers which have bound non-spe-

cifically to template DNA [4], thereby generating consis-

tently high PCR specificity.

The transgenic pigs have been generated for preventing

xenograft rejection [21]. It cannot be ruled out that the

integration of the transgene may be into or adjacent to the

locus of a PERV provirus, potentially leading to an enhanced

virus expression. Therefore, our research also concentrated

on differences in the copy number of PERV subtypes

between transgenic pigs. Our results did not reveal statisti-

cally significant differences in the copy number of PERVs

between pigs expressing the human a-1,2-fucosyltransferase

and human aGal genes, and pigs with both genetic modifi-

cation, which remains in agreement with other findings.

Dieckhoff et al. [8] postulated that the expression of PERVs

was not different between transgenic pigs and non-trans-

genic animals and indicated that differences in PERV

expression correlated with the genetic background of the

animals, not with the specific transgene.

Currently, we could not find any published reports

regarding the identification of correlation between the

gender of the pigs and the copy number of PERVs.

According to the our opinion, the amount of PERVs is not

dependent on animal gender. This may suggest that PERV

proviruses are integrated into autosomes, rather than into

sex chromosomes.

In conclusion, the quantitative analysis revealed differ-

ences in the copy number of PERV subtypes between

various organs of transgenic pigs generated for xeno-

transplantation. Our data support the idea that careful pig

selection for organ donation with low PERV copy number

may limit the risk of retrovirus transmission to the human

recipients.

Acknowledgments This study was supported by the project no. NR

12 0036 06, which was financed from 2009 to 2013 by the National

Centre for Research and Development in Poland.

Conflict of interest The authors declare that there are no conflicts

of interest.


123

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