Short- and long-term skin graft survival in cattle clones with different mitochondrial haplotypes

Short- and long-term skin graft survival

in cattle clones with different

mitochondrial haplotypes

Christine L. Theoret a,*, Monique Dore b, Pierre-Yves Mulon c,Andre Desrochers c, Francisco Viramontes d, France Filion d,

Lawrence C. Smith a,d

a Departement de biomedecine veterinaire, Faculte de medecine veterinaire, Universite de Montreal,

3200 rue Sicotte, Saint-Hyacinthe, Quebec, Canada J2S 7C6b Departement de pathologie et microbiologie, Faculte de medecine veterinaire,

Universite de Montreal, Saint-Hyacinthe, Quebec, Canada J2S 7C6c Departement des sciences cliniques, Faculte de medecine veterinaire, Universite de Montreal,

Saint-Hyacinthe, Quebec, Canada J2S 7C6d Centre de recherche en reproduction animale, Faculte de medecine veterinaire,

Universite de Montreal, Saint-Hyacinthe, Quebec, Canada J2S 7C6

Received 11 May 2005; accepted 17 August 2005

Abstract

In contrast to nuclear DNA, cytoplasmic genes may differ among cloned animals due to the presence

of polymorphic mitochondrial DNA haplotypes in the host oocytes, raising doubts about histocompat-

ibility among clones. Three bovine clones were generated by nuclear transfer; dermal fibroblasts from a

fetus were used as donor cells, whereas oocytes from abbatoir-derived ovaries were used as recipient

cells. The mitochondrial DNA (sequencing of coding and non-coding regions) and nuclear DNA (13

microsatellite markers) of cloned and control animals were characterized to identify potential

polymorphisms. Skin auto- and allografts were transplanted on the adult clones and a non-related

animal as a measure of immunological reactivity. Nuclear DNA of cloned animals was genetically

identical but differed in all microsatellites of the non-related control. Amounts of donor cell

mitochondrial DNA in the skin ranged from 1 to 2.6% among clones. Few differences in heteroplasmy

were observed between skin and WBC of the clones, indicating limited mitochondrial DNA segregation

www.journals.elsevierhealth.com/periodicals/the

Theriogenology 65 (2006) 1465–1479

* Corresponding author. Tel.: +1 450 773 8521x8517; fax: +1 450 778 8109.

E-mail address: [email protected] (C.L. Theoret).

0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2005.08.019

in tissues during pre- and post-natal development to adulthood. Sequencing of the remaining oocyte-

derived mitochondrial DNA haplotype identified polymorphisms in coding and non-coding regions,

confirming their origin from unrelated maternal lineages. Nonetheless, skin transplants between clones

were accepted for the 92 d study period, whereas third-party grafts were rejected. In conclusion, the

nuclear transfer-generated adult bovine clones used in this study were immunologically compatible

with one another despite differences in their mitochondrial DNA haplotypes.

# 2005 Elsevier Inc. All rights reserved.

Keywords: Cloning; Nuclear transfer; Polymorphism; Histocompatibility; Bovine

1. Introduction

Nuclear transfer (NT) technology re-programs a somatic cell to generate embryos for

isolation of either genetically superior intact animals (reproductive cloning) or embryonic

stem (ES) cells for regenerative therapy (therapeutic cloning). Although in principle, the

products of NT should be genetically identical to the parental somatic cell in terms of

nuclear DNA (nDNA), it has been shown that first-generation cloned animals are not

identical in terms of gene expression (reviewed by [1]). Although errors in the epigenetic

reprogramming of nDNA have often been blamed for the variability among clones, this

may relate to an incompatibility between nDNA and mitochondrial DNA (mtDNA) from

the donor and mtDNA inherited from the recipient oocyte [2–5]. Indeed, nuclear-

mitochondrial incompatibilities may also be partially responsible for the high embryonic

mortality and neonatal morbidity of most animal clones.

When NT is used for animal cloning, donor cell cytoplasm containing mtDNA is

introduced together with the nDNA-containing nucleus into the recipient oocyte. It has

been proposed that the mitochondria transmitted by somatic cell NT may be responsible for

observed phenotypic changes in cloned animals [6]. Indeed, due to the role mitochondria

play in generating cellular ATP, mitochondrial sequence polymorphisms have been

reported to affect growth and physical performance in mice [7], milk quality in Holstein

cows [8], carcass traits in Japanese Black cattle [9] and fertility in beef cattle [10].

However, mitochondrial gene products seem also to modulate tissue immunogenicity, a

role much less understood to date [11,12]. Nonetheless, a recent study showed that skin

transplants between genetically identical cloned pigs were accepted; however, differences

in mtDNA between the clones were not considered [13].

Therapeutic cloning could provide a limitless source of cells for regenerative therapy

and obviate the need for immunosuppression required in conjunction with most

transplantation procedures. The success of therapeutic cloning theoretically depends on the

immunological compatibility of the products generated by NT. A recent study performed in

cattle found that somatic cell NT-generated ES cells bioengineered into tissues were not

rejected and remained viable after transplantation into the nuclear donor animal, even

though they expressed a different mtDNA haplotype [14].

The main goal of this study was to assess short- and long-term immunological reactivity

of cloned tissues, as a measure of phenotypic identity in somatic cell NT-derived bovine

clones. Skin grafting was used for this purpose, as grafts are obtained easily and with

C.L. Theoret et al. / Theriogenology 65 (2006) 1465–14791466

minimal morbidity, and because skin is the most immunogenic of all transplantable foreign

organs and tissues. Moreover, we wished to correlate the response to grafting to the

presence of polymorphic mtDNA haplotypes.

2. Materials and methods

2.1. Production of somatic cell cloned calves

All procedures were performed in compliance with the Guide for the Care and Use of

Agricultural Animals in Research and Training, by the Universite de Montreal’s guidelines

and were sanctioned by the Canadian Council on Animal Care [15]. The clones were

produced by somatic cell NT following standard procedures [16]. Briefly, fibroblasts from

a 60-d-old fetus were used as donor cells. The fetus was produced by artificial insemination

of a Holstein heifer (Bos taurus) carrying a mtDNA mutation, with semen of a Zebu bull

(Bos indicus). The mtDNA mutation consisted of a 63 base pair addition in the D-loop, that

was used to trace the donor mtDNA in the clones. Oocytes of Holstein cows derived from

slaughterhouse ovaries and carrying wild-type mtDNA were used as recipient cells. Donor

fibroblasts were fused to enucleated oocytes and cultured in vitro for 7 d in a humidified

atmosphere of 5% O2, 5% CO2 and 90% N2. After in vitro culture, blastocyst-stage

embryos were transferred to the uterus of estrus-synchronized recipient heifers and allowed

to develop to term. Cloned offspring were delivered by Caesarean section at 290 d of

gestation and housed indoors thereafter. The clones were delivered by Caesarean section to

diminish the risk of parturition problems. There were only three clones in this experiment

and all were born with normal weights and in perfect health. Therefore, there was no

selection since all three animals were used in our experiment.

2.2. Nuclear and mitochondrial DNA analysis

Total genomic DNA was extracted from skin biopsies and blood of cloned and control

animals using standard procedures (DNeasy Kit, Qiagen Inc., Mississauga, ON, Canada).

The genetic identity of the nDNA of cloned animals was determined by microsatellite

analysis using 12 microsatellite markers (Bova-Can Laboratories, Saskatoon, SK, Canada).

The sequencing of mtDNA was obtained by PCR amplification of three regions

encompassing approximately 20% (3217 out of 16338 base pairs) of the entire bovine

mitochondrial genome. The mtDNA coding regions analyzed included the genes for mt-Nd1

(mitochondrial NADH dehydrogenase 1; primers: ND1-3020F, 50-CTTAGTTAAGGTGG-

CAGAGC-30 and ND1-4147R, 50-GTAGGTTCGATTCCTATAGTTC-30), mt-Atp6 (mito-

chondrial ATP synthase 6; primers: ATP6-8099F, 50-GATTGAGAGCCATATACTCTC-30

and ATP6-9033R, 50-AAGCTCCTGTAAGAGGTCAAG-30), mt-Cytb (mitochondrial

cytochrome b), mt-Tt (mitochondrial tRNA threonine), mt-Tp (mitochondrial tRNA

proline) and the entire non-coding D-loop region (primers: CytB-F, 50-CTACTATACCAT-

TAAGGACATC-30 and MT12S-R, 50-GCATTTTCAGTGCCTTGCTTTG-30). The PCR

products were ligated into a pDrive cloning vector (Qiagen) and DH5 a competent cells

(Invitrogen) and were transformed with the resulting plasmid following the manufacturer’s

C.L. Theoret et al. / Theriogenology 65 (2006) 1465–1479 1467

protocol (Qiagen PCR Cloning Kit). Positive colonies were expanded overnight in liquid

Luria-Bertani medium and DNA was extracted and purified (Qiaprep Spin Miniprep Kit,

Qiagen) to obtain sufficient quantities for sequencing. Sequencing reactions were performed

on PCR clones via the dideoxy sequencing method (big dye terminator 3.0; ABI prism;

Applied Biosystems, PE, Branchburg, NJ, USA) using the Sp6 and T7 oligos (1 mM).

Sequencing reactions of mtDNA clones were analyzed (Sequencher Software, Version 3.1.1,

Ann Arbour, MI, USA) to identify polymorphism when compared to the Bos taurus mtDNA

sequence [17].

2.3. Quantification of donor cell-derived mtDNA

A region of the mtDNA D-loop containing the mutation (63 base pair addition) was

amplified by PCR (primers: 15913Bov-F, 50-CATAACACGCCCATACAGAC-30 and

16176BOV-R, 50-TAGCGGGTTGCTGGTTTCAC-30) under the following conditions: 40

cycles of 94 8C for 30 s, 56 8C for 30 s and 72 8C for 30 s using 50 ng of total DNA

extracted from skin and blood of cloned and control animals. Following spectro-

photometric quantification, 1 ng of the first PCR product was used for a second nested

PCR reaction (primers: 15938Bov-F, 50-AGAATGAATTACCTACGCAAG-30 and

16140Bov-R, 50-GCTCGTGATCTAATGGTAAG-30), under the following conditions:

30 cycles of 94 8C for 30 s, 60 8C for 30 s and 72 8C for 30 s. PCR amplifications resulted

in fragments of 202 base pairs (wild-type) and 268 base pairs (mutant), which were

sequenced to confirm mtDNA origin. A standard curve was generated using serial

dilutions of mutant in wild-type mtDNA, which was amplified by PCR and run on

a 1.5% agarose gel together with the PCR samples from the cloned animals (Fig. 1).

Percentage of mutant mtDNA was determined by image analysis of the UV-

transilluminated ethidium bromide-stained gel using the integrated density values

(IDV) obtained by photometric software (Alpha-Imager 2200, Alpha Innotech, San

Leandro, CA, USA). Eight gels, each with standard curves with a 0.98 coefficient of

determination (R2), were analyzed to determine mutant mtDNA ratio values in tissues of

cloned and control animals.

2.4. Skin grafting and biopsy procedures

Skin grafting was performed between the three 18-mo-old male bovine clones (animals

A, B and C) and one unrelated 12-mo-old Holstein bull (animal D) with no

immunosuppressive therapy. Each animal received an autograft, as well as an allograft

from each of the three other individuals, in quadruplicate, which were placed in four

separate locations (zones 1–4) on its back (Fig. 2).

Following local anesthesia, 8 mm diameter, full-thickness skin grafts were obtained

laterally within the designated zone, whereas 6 mm recipient sites were created

medially. The 8-mm grafts were press-fitted within the appropriate 6-mm recipient bed,

with hair follicles reversed to facilitate further observations. The grafts were held in

place with n-butyl cyanoacrylate glue (3 M Vetbond tissue adhesive 1469; London, ON,

Canada) then dressed. Bandages were inspected daily and changed twice during the 7-d

period following surgery. Graft survival was evaluated macroscopically and documented


photographically at each bandage change. Visual and digital inspection allowed

evaluation of the following criteria: edema (protrusion of graft above surrounding skin

edges); contraction (separation of graft margins from surrounding skin); dehydration

(hardness to the touch); sloughing; hair growth; presence of granulation tissue; and

presence of a shiny, hairless scar.

Eight-mm full-thickness punch biopsies of the two most cranial grafts from each

location in each animal were obtained 1 wk post-operatively. Biopsy specimens included

4 mm of the host skin and 4 mm of the graft. The remaining grafts were left unbandaged

and observed bi-weekly until they were biopsied, as described above, 13 wk post-


Fig. 1. (A) Representative agarose gel showing the PCR products of a serial dilution of mutant (268 bp) and wild-

type (202 bp) mtDNA. (B) Standard curve (linear regression) and coefficient of determination (R2) value of the

eight gels used to quantify the percentage of mutant mtDNA in tissues. Circles identify the position of the average

amount of mutant mtDNA in each clone. (C) Representative agarose gel used to quantify the percentage of mutant

mtDNA in the skin and white blood cells (WBC) of cloned (A–C) and control (D) animals.

operatively. Tissue specimens were immersed in 10% buffered formalin overnight at room

temperature, then embedded in paraffin.

2.5. Microscopic assessment

Histological sections, stained with hematoxylin eosin phloxin saffron, included three

distinct areas: adjacent host skin, junction between host skin and graft and the graft. In

each, the epidermis was described as normal, necrotic, hyperplastic or sloughed, whereas

the superficial, mid and deep dermis were separately assessed for inflammatory cellular

infiltrate (neutrophils, lymphocytes, plasma cells, macrophages, eosinophils), edema,

hemorrhage, fibrin exudate, fibrosis and the number and state of blood vessels and dermal

appendages. Evaluations were performed, in the blind, by two independent observers.

Special attention was paid to migration of host epithelium under graft edges, perivascular

macrophages in surrounding tissues and thickening of the arteriolar wall of graft vessels.

2.6. Statistical analysis

Statistical analysis of the ratio of donor cell-derived mtDNA in skin and white blood cell

(WBC) from clones was performed by ANOVA and the tissue and animal means were

compared by the Tukey–Kramer multiple-comparison test.


Fig. 2. Template for skin grafting procedure.

3. Results

3.1. Nuclear-derived DNA

Microsatellite analysis of the nDNA extracted from the clones confirmed the clonal

identity among the three clones and the fetal-derived donor fibroblast cell line used for NT

(Table 1). Moreover, the large majority of the 13 microsatellite markers employed differed

among clones and the animal used as a non-related control in these experiments, suggesting

no possible relationship among the control and the clones.

3.2. Ratio of donor cell- and oocyte-derived mtDNA (heteroplasmy)

The ratio of donor-derived mtDNA present in the skin and WBC of the cloned animals

was assessed to determine whether the levels of self mtDNA varied significantly among

clones (Table 2). The average quantities of mtDNA remnant from the donor cell was quite

low in the tissues analyzed, indicating that the non-self mtDNA originating from the host

oocyte comprised over 95% of the mitochondrial haplotypes present in the clones. Few

differences in heteroplasmy were observed between skin and WBC of the clones,

indicating limited mtDNA segregation in tissues during pre- and post-natal development to

adulthood. Nonetheless, clone B had slightly higher amounts of donor-derived mtDNA

when compared to clones A and C, suggesting that mtDNA segregation patterns may vary

slightly among individual clones. Therefore, donor cell-derived mitochondrial haplotypes

comprised only a minute proportion of the total mtDNA present in the skin and WBC of the

three clones used in these experiments.

3.3. Sequencing and characterization of mtDNA haplotypes

We sequenced three regions of the mtDNA comprising the genes for mt-Nd1 (nt3099 to

nt4056), mt-Atp6 and 8 (nt8128 to nt9753), and the genes for mt-Cytb (partial), mt-Tt and


Table 1

Microsatellite analysis of nDNA from clones and control animals and of the donor cell line used for nuclear

transfer

Microsatellite markers Clone A Clone B Clone C Control D Donor cell

BM1824 182/192 182/192 182/192 178/180 182/192

BM2113 135/135 135/135 135/135 135/137 135/135

CSSM36 179/179 179/179 179/179 163/179 179/179

ETH10 211/213 211/213 211/213 221/225 211/213

ETH225 150/158 150/158 150/158 148/150 150/158

ETH3 103/127 103/127 103/127 117/117 103/127

HEL1 106/110 106/110 106/110 112/112 106/110

INRA0123 202/214 202/214 202/214 200/206 202/214

SPS115 246/248 246/248 246/248 248/252 246/248

TGLA122 143/163 143/163 143/163 143/163 143/163

TGLA126 117/121 117/121 117/121 115/117 117/121

TGLA227 77/91 77/91 77/91 93/97 77/91

mt-Tp as well as the entire control region, D-loop (nt15204 to nt363). When analyzing part

of the mtDNA sequence (D-loop, two tRNAs and four mRNAs) for the donor cell, the three

adult clones and the control animal, a few deletions (clone B) and some point mutations

(clones A, B and control D) were found, suggesting that the animals contained mtDNA

from different maternal lineages (Table 3). The animals were similar in the few coding

regions that were analyzed, which showed two silent point mutations in mt-Atp6 (clone A)

and mt-Nd1 (clone B). However, one exception was a T to C mutation in the mt-Cytb gene

of clone B, which could be a potential source of histocompatibility antigens. The mtDNA

from the fetus used to derive fibroblasts for NT contained both additions and point

mutations in the D-loop but no polymorphism in the coding regions of the mtDNA

analyzed.


Table 2

Mean (�S.E.M.) percentages of donor-derived mtDNA (heteroplasmy) in the skin and white blood cells (WBC) of

adult clones at 18 mo of age

Skin WBC Clone mean

Clone A 0.71 a � 0.25 0.99 ab � 0.25 0.85 A � 0.18

Clone B 2.28 c � 0.25 3.55 d � 0.25 2.92 B � 0.18

Clone C 1.50 abc � 0.25 1.34 abc � 0.25 1.42 A � 0.18

Tissue mean 1.50 A � 0.14 1.96 B � 0.14 1.73

Different lower case letters denote significant differences in the Tukey–Kramer multiple-comparison test.

Different upper case letters denote significant differences among either tissue or clone groups.

Table 3

Nucleotide localization of sequence variations in the mtDNA haplotypes of cloned and control animals and the

fetal fibroblast donor cells used for NT

mtDNA region Mutation positiona Clone A Clone B Clone C Control D Donor cell

mt-Nd1 3130 – T to C – – –

mt-Atp6 8949 A to C – – – –

mt-Nd8 None – – – – –

mt-Cytb 15399 – T to Cb – – –

mt-Tp None – – – – –

mt-Tt None – – – – –

D-loop 15973-94 – – – – Additionc

16074 – – – – T to C

16130 – T deletion – – –

16136-7 – 2T deletion – – –

16141 – T to C – – –

16231 C to T – – – –

12 – – – – A to G

169 – A to G – A to G A to G

173 A to G – – A to G –

221 – – – – 2C addition

a Nucleotide positions according to [16].b Nucleotide substitution leading to an amino acid substitution (phenylalanine to serine).c Addition of three repetitive sequences of 21 base pairs (i.e. a 63 base pair addition).

3.4. Macroscopic observations of skin grafts

None of the grafts were rejected in the acute period; rather, autografts and clone grafts

remained flush with the surrounding host skin, whereas allografts showed a moderate

amount of contraction and protruded slightly. By 3 wk post-operatively, some allografts in

the control animal had been rejected and were replaced by granulation tissue within the

recipient bed as well as migrating epithelium at the border. Although allografts in the

clones were still in place, contraction of the graft was maximal and surface epithelium had

begun to slough. Conversely, new hair growth was observed in most autografts at this time

strongly indicating graft acceptance. All allografts were rejected by 4 wk in the control

animal, whereas autografts (technical control) were accepted for the duration of the study

(13 wk). In the clones, allografts were rejected within 6 wk and were replaced by shiny,

hairless scar tissue. Conversely, both autografts and grafts from the other clones were

accepted for the duration of the study (Fig. 3A–C).

3.5. Histological observations of skin grafts during the acute period (1 wk)

3.5.1. AutograftsThe epidermis was normal in appearance and continuous between adjacent host skin

and the graft, although a narrow band of immature granulation tissue was present at this


Fig. 3. (A) Autograft and (B) clone graft in clone C, 6 wk post-operatively. Hair growth is indicative of graft

acceptance. (C) Allograft in clone B, 6 wk post-operatively. Shiny scar tissue has replaced the rejected graft.

junction at the level of the superficial dermis in the control animal (Fig. 4A). Cutaneous

appendages were intact and none of the dermal zones within the graft or adjacent host

tissue displayed inflammation, edema, hemorrhage or fibrin exudate.

3.5.2. Clone graftsThe epidermis was continuous at the junction between host skin and graft, and displayed

only a mild diffuse hyperplasia with occasional serocellular crusts (Fig. 4B). The superficial

dermis showed very little inflammation, although a narrow band of immature fibrous tissue

was present at the junction between host skin and graft in two cases. Middle and deep dermis

of the graft showed a few fibrotic foci that were occasionally infiltrated by a small number of

eosinophils and lymphocytes. Overall, clone grafts were indistinguishable from autografts.

3.5.3. AllograftsHost epidermis adjacent to the graft presented a moderate diffuse hyperplasia, while the

graft epidermis was completely necrotic and infiltrated by a large number of both intact and

degenerate neutrophils. The junction between host skin and graft was either discontinuous

or the host epidermis had begun migrating under the graft. A severe inflammatory response,


Fig. 4. Photomicrograph of 1-wk-old (acute) graft sites in clone A. (A) Autograft and (B) clone graft. Continuous

epidermis between adjacent host skin and graft; band of immature granulation tissue present at the host-graft

junction (delineated by dotted line). (C) Allograft. Host epidermis presents moderate diffuse hyperplasia

(arrowheads); massive necrosis is evident in all layers of graft, consistent with acute cellular rejection.

Bar = 100 mm.

consisting mainly of large perivascular infiltrates of lymphocytes and eosinophils, was

present in the superficial dermis of the host skin, whereas the superficial dermis of the graft

was severely infiltrated by eosinophils. Many necrotic blood vessels accompanied by a

fibrinous exudate were noted in the grafts’ superficial dermis. Focally extensive areas of

fibrosis were present at the dermal junction between host skin and graft. All cutaneous

appendages in the grafts were necrotic. Vasculitis, thrombotic vessels, exudation of fibrin

and hemorrhage were observed in the deep dermis, particularly within the graft, along with

an important zone of fibrosis infiltrated by eosinophils, lymphocytes and neutrophils below

the graft bed. The overall picture was consistent with acute cellular rejection (Fig. 3C).

3.6. Histological observations of skin grafts during the chronic period (13 wk)

3.6.1. AutograftThe epidermis was continuous between host skin and graft, and the superficial dermis in

all grafts included normal hair follicles, sebaceous and sweat glands, although in reduced

numbers compared to adjacent host skin (Fig. 5A). In some cases, a narrow band of fibrous

tissue was present at the junction between host skin and graft.


Fig. 5. Photomicrograph of 13-wk-old (chronic) graft sites. (A) Autograft and (B) clone graft in clone A.

Continuous epidermis between host skin and graft; presence of dermal appendages in graft; narrow band of fibrous

tissue at host-graft junction (delineated by dotted line). (C) Allograft site in clone B. Fibrotic zone covered by thin

epidermis and devoid of cutaneous appendages, consistent with second-intention repair following graft rejection.

Bar = 100 mm.

3.6.2. Clone graftsAside from a reduced number of sweat glands, the clone grafts were comparable to

autografts 13 wk post-transplantation (Fig. 5B).

3.6.3. AllograftsThe area where the graft had been was replaced by a wide triangular zone of fibrosis

covered by thin epidermis and completely devoid of dermal appendages (Fig. 5C). In some

cases, lymphocytic infiltrates were present beneath the area of fibrosis. These observations

were consistent with scar tissue generated by second-intention repair following complete

graft rejection.

4. Discussion

The purpose of this investigation was to assess immunogenicity of cloned tissues, in an

effort to estimate phenotypic identity in NT-derived bovine clones. While it has been

proposed that the mitochondria transmitted from the host oocyte during NT may

determine, in part, the phenotype of cloned animals, the level of inheritance of donor cell

mtDNA within somatic cell clones generated by this technology remains unclear. Most

studies have found little or no mtDNA from the donor cell in animals derived by somatic

NT [18–20], supporting the view that somatic cell clones carry nearly exclusively the

mtDNA originating from the host oocyte. Conversely, other studies have reported that

some clones may contain as much as 60% of the donor cell mtDNA haplotype [6].

Therefore, it is important to accurately quantify the level of mtDNA heteroplasmy in

experiments aimed at examining the effect of mtDNA haplotype on the phenotypic

variability among cloned animals.

We used fibroblast donor cells carrying a mutation consisting of a 66 base pair insertion

in the D-loop of the donor mtDNA, which enabled straightforward and secure discernment

from the recipient oocyte mtDNA, to establish the level of inheritance. When analyzing

part of the mtDNA sequence (D-loop, two tRNAs and four mRNAs) of the donor cell, the

three adult clones and the control animal, a few deletions (clone B) and some point

mutations (clones A, B and D) were found, suggesting that the host oocytes used to produce

the different clones came from females of different maternal lineages. The animals were

likewise similar in the few coding regions that were analyzed, that showed two silent point

mutations in mt-Atp6 (clone A) and mt-Nd1 (clone B). However, one exception was a T to

C mutation in the mt-Cytb gene of clone B. Regardless of this amino acid substitution, the

degree of macro- and microscopic graft acceptance in both short- and long-term

evaluations was similar among all three clones, indicating that the change from

phenylalanine to serine in mt-Cytb did not render clone B less compatible in comparison to

clones A and C.

Both class I and II major histocompatibility (MHC) antigens, responsible for eliciting

the strongest rejection response, are determined by nDNA. On the other hand,

maternally-inherited mtDNA is capable of generating endogenous peptides that could

stimulate a T-cell response specific for mtDNA-encoded minor histocompatibility

antigens (miHA)s. Mitochondrially transmitted histocompatibility antigens remain


poorly understood in most species, particularly in cattle where no previous reports have

addressed this question. In order for genetic polymorphisms to give rise to a functional

miHA, they must generate at least two foreign peptides: one capable of presentation

by a MHC class II molecule (to generate a helper determinant for CD4+ T-cells) and

one capable of presentation by a MHC class I molecule (to generate a determinant for

CD8+ T-cells) [21]. Skin graft rejection has been shown in inbred homozygous mice

carrying different mtDNA [22]. Specifically, the maternally transmitted antigen (MTA)

of mice is a model miHA consisting of a peptide (MTF) that is presented on the cell

surface by an H-2 class I molecule, HMT. In mice, MTF is derived from mt-Nd1, a

mitochondrially encoded protein, and the amino-terminal N-formyl-methionine is

essential for binding to HMT [23]. Unlike mouse MTA, the rat MTA depends on a

classical class I molecule for its expression, which is derived from a peptide originated

from an internal region of the mt-Atp6 mitochondrial gene [12]. Due to the diversity

between mouse and rat MTAs, it is plausible to speculate that, apart from mt-Nd1 and

mt-Atp6, polymorphisms present in other coding regions of the mtDNA, including the

mt-Cytb gene, could also instigate MTAs that compromise graft compatibility in bovine

and other mammals.

The present study has shown that cattle produced by NT inherit the majority of their

mtDNA from the recipient oocyte rather than from the donor cell. In spite of this, it appears

that the presence of non-self mitochondrial proteins in cloned bulls does not lead to

immunogenicity after tissue transplantation. Indeed, despite the fact that cloned cattle are

capable of mounting an immune response and cloned cattle grafts are immunogenic, we

showed that genetically identical adult cattle clones are immunologically compatible. Our

experimental model precluded offspring-to-parent transplants because the parent cells

came from a 60-d fetus. However, offspring-to-offspring clone transplants are likely to be a

close approximation because both carry identical nDNA and must interact with potential

non-self mtDNA MTA. In this study, skin transplants were used as a measure of phenotypic

identity, since skin is the most immunogenic of all transplantable organs and tissues, with

one-half of its cells being of immunological origin [24]. We based the 13 wk study period

on a previous investigation showing that skin grafts exchanged between chimeric co-twin

calves produced by embryo transfer survived longer than grafts between unrelated animals,

but were always rejected within 10 wk of transplantation [25]. A recent study confirmed

that skin transplants between genetically identical cloned pigs were accepted; however, the

histological status of the grafts was only followed for 17 d post-transplantation [13]. While

our findings corroborate the former, given that we observed allograft rejection up to 6 wk

post-operatively, late graft rejection might have been overlooked in the aforementioned

study.

Our data concurred with previous studies showing that NT-generated bovine fetuses

yielded ES cells and bioengineered tissues that were immunologically compatible with the

nuclear donor animal, even though they expressed a different mtDNA haplotype [14].

Despite the potential immunogenicity of the amino acid substitution we found in the mt-

Cytb gene of clone B, skin grafts survived the duration of the experiment, possibly as a

result of the small number of novel peptides generated from mitochondrial haplotypes

present in the clones, or because the particular genetic polymorphisms we detected might

not lead to the expression of miHAs. It is currently impossible to predict the impact of


amino acid substitutions on the ability of mtDNA-encoded peptide either to bind to bovine

class I MHC molecules or to activate cytotoxic T lymphocytes [14].

In conclusion, our data, combined with those obtained by other investigators, suggest

that even multiple mtDNA genetic polymorphisms might not lead to the expression of

miHAs in a particular donor–recipient combination, whereas they might in others. In

practice, this implies that histocompatible tissues can be generated using the NT approach

in cattle, despite the presence of oocyte polymorphic mitochondrial genes. Nonetheless, as

would be true for any species, cattle mitochondrial haplotypes that were not part of the

present trial may be sufficiently immunogenic to cause graft rejection. Therefore, to

ascertain whether therapeutic cloning is viable for human applications, further grafting

experiments between animal clones carrying known MTA polymorphisms, would be

required to confirm that the immune compatibility is complete, regardless of their mtDNA.

In the meantime, careful screening of potential MTA hotspots, currently mt-Nd1 and mt-

Atp6 genes, in the mtDNA of both the cloned-derived ES cell lines and human hosts, is

advised.

Acknowledgement

Research funded by the Canada Research Chair Program (LCS is Chair in Animal

Cloning and Embryo Biotechnology).

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Short- and long-term skin graft survival in cattle clones with different mitochondrial haplotypes

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