Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
204 Poster Abstracts<br />
Conclusions These prelim<strong>in</strong>ary data suggest that flow cytometry<br />
could be used for evaluat<strong>in</strong>g sperm DNA b<strong>in</strong>d<strong>in</strong>g capacity. Besi<strong>de</strong>s<br />
this study shows that un<strong>de</strong>r our experimental conditions the capacity<br />
of b<strong>in</strong>d<strong>in</strong>g of DNA to bull frozen-thawed bull spermatozoa is related<br />
to viable and unviable cells. These facts ma<strong>in</strong>ta<strong>in</strong> opened the<br />
possibility of us<strong>in</strong>g this technique <strong>in</strong> IVF systems for generation of<br />
transgenic bov<strong>in</strong>e embryos.<br />
Supported by BIOCARM 10BIO2005/01-6463 and MEC-FEDER<br />
AGL2006-03495<br />
P534<br />
On the role of LIF and LIFR expression dur<strong>in</strong>g rabbit<br />
embryonic stem cell l<strong>in</strong>e establishment<br />
Gócza, E.*, Catunda, AP., Hiripi, L., Bősze, Zs.<br />
Agricultural Biotechnology Center, 2100, Szent-Györgyi A. str. 4, Gödöllő,<br />
Hungary<br />
Embryonic stem cells (ES) have become an important tool for<br />
generat<strong>in</strong>g transgenic mice. Embryonic like stem cells and ES<br />
chimeric animals have been reported for several farm animal species,<br />
but germ l<strong>in</strong>e transmission was not reported.<br />
Rabbit ES cells, established <strong>in</strong> our laboratory, by their growth <strong>in</strong><br />
tightly packed, flat colonies resembled to human ES cell l<strong>in</strong>es, while<br />
expression of alkal<strong>in</strong>e phosphatase, Oct-4, Nanog, SSEA-1 and CD9<br />
which were <strong>de</strong>tected by immunosta<strong>in</strong><strong>in</strong>g ma<strong>de</strong> them similar to mouse<br />
ES cells.<br />
Leukemia <strong>in</strong>hibitory factor (LIF) is necessary for mouse ES cell selfrenewal,<br />
but it fails to ma<strong>in</strong>ta<strong>in</strong> self-renewal <strong>in</strong> human and non-human<br />
primate ES cells. To exam<strong>in</strong>e the role of LIF and leukemia <strong>in</strong>hibitory<br />
factor receptor (LIFR) at rabbit ES cell l<strong>in</strong>e establishment, the<br />
expression patterns of LIF and LIFR were analysed at different stages<br />
of rabbit embryonic <strong>de</strong>velopment and LIFR expression <strong>in</strong> rabbit ES<br />
cells by RT-PCR at different passages. LIFR mRNA was first<br />
<strong>de</strong>tected around implantation <strong>in</strong> 6.5 d.p.c. rabbit embryos, while LIF<br />
mRNA expression was already present at rabbit blastocyst stage. In<br />
attached ICM and <strong>in</strong> ES cells after the 1st passage, very low level of<br />
LIFR expression was found. After the 2nd passage high expression of<br />
LIFR was <strong>de</strong>tected <strong>in</strong> rabbit ES cells.<br />
S<strong>in</strong>ce commercial rabbit LIF is not available we had to f<strong>in</strong>d the most<br />
effective recomb<strong>in</strong>ant LIF for rabbit ES cell establishment. With this<br />
object human, rat and mouse recomb<strong>in</strong>ant LIFs were compared. At<br />
early passages we could not f<strong>in</strong>d any difference <strong>in</strong> their effect, but at<br />
higher passage numbers, the rat LIF was found to be the most efficient<br />
factor <strong>in</strong> the prevention of the differentiation. LIF withdrawal resulted<br />
the differentiation of rabbit ES like cells <strong>in</strong>to cardiomyocytes. These<br />
f<strong>in</strong>d<strong>in</strong>gs suggest that the the LIF-LIFR signal pathway has an<br />
important role <strong>in</strong> ma<strong>in</strong>tenance of pluripotency of rabbit ES cells.<br />
This work was supported by grant OM-00367/2004, OTKA T037582<br />
and Bilateral Intergovernmental S and T Cooperation D-26/02<br />
(HUN02/045).<br />
P535<br />
Succesful h-lactoferr<strong>in</strong> transgenic goat embryo transfer<br />
after SCNT<br />
Mutto, A 1 * # ; Kaiser, GG 2# ; Mucci, N 2# ; Hozbor, F 2 ; Sanchez, E 2 ; Ugal<strong>de</strong>, R 1 ;<br />
Alberio, RH 2<br />
1IIB, UNSaM, Argent<strong>in</strong>a; 2 INTA Balcarce, Argent<strong>in</strong>a<br />
#Authors contributed equally to the present work<br />
Because of its properties (digestibility, composition, taste), goat milk<br />
is suitable to be used as a substitute for cow milk <strong>in</strong> patients with cow<br />
milk allergy and for human nursl<strong>in</strong>g babies with no tolerance to<br />
lactose. By add<strong>in</strong>g some sugars, lisozime C and lactoferr<strong>in</strong> it will be<br />
possible to have a “humanized” goat milk.<br />
The objective of this work was to produce transgenic clone embryos<br />
and pregnancies for human lactoferr<strong>in</strong> <strong>in</strong> goats by us<strong>in</strong>g somatic cell<br />
nuclear transfer of transfected fetal goat fibroblasts.<br />
Materials and methods A 1960bp human lactoferr<strong>in</strong> cDNA (Gen<br />
Bank n: NM_002343) obta<strong>in</strong>ed from ATCC encompass<strong>in</strong>g the entire<br />
cod<strong>in</strong>g region was used as template. It was cloned <strong>in</strong>to pBC1 milk<br />
expression vector (Invitrogen, Ca, USA) between the <strong>in</strong>tron 1, exon 2<br />
and exon 7 of goat β-case<strong>in</strong> gene. An eukaryotic selection marker<br />
cassette with a Blasticid<strong>in</strong>-S resistence gene was ad<strong>de</strong>d, un<strong>de</strong>r h-EF1α<br />
promotor.<br />
Goat fetal fibroblasts obta<strong>in</strong>ed from 30 day pregnancies were sexed<br />
by PCR and karyotyped and female cell l<strong>in</strong>es were transfected by<br />
liposomes (Lipofectam<strong>in</strong>e 2000®, Invitrogen, USA). Colonies were<br />
selected after 10 days of culture with Blasticid<strong>in</strong> and grown <strong>in</strong><br />
complete culture media (D-MEM). Stable transfections were<br />
confirmed by FISH.<br />
Oocytes were obta<strong>in</strong>ed by laparoscopic ovum pick-up of stimulated<br />
goats dur<strong>in</strong>g the reproductive season, <strong>in</strong> vitro matured and subjected<br />
to nuclear transfer as <strong>de</strong>scribed by Baldassarre et al (2004).<br />
Presumptive zygotes (24 hs after NT) were transfered by a surgical<br />
procedure <strong>in</strong> both oviducts of those animals that presented at least 2<br />
hemorrhagic corpora lutei.<br />
Results A total number of 331 oocytes were recovered from 480<br />
follicles after 5 sessions of LOPU (8 goats each session). All oocytes<br />
that presented a polar body were enucleated and transferred (n=238).<br />
There was an overall fusion rate of 51%. A total number of 121<br />
presumptive zygotes were transfered to 12 goats (7 to 12 per<br />
recipient), result<strong>in</strong>g <strong>in</strong> 5 pregnancies at day 30 (41.6% pregnancy rate)<br />
and 2 at day 83 (16.6%). Two pregnancies were tw<strong>in</strong>s, one rema<strong>in</strong><strong>in</strong>g<br />
until day 83.<br />
We can conclu<strong>de</strong> that un<strong>de</strong>r our conditions it is posible to obta<strong>in</strong><br />
transgenic goat fetal fibroblasts, embryos and pregnancies for human<br />
lactoferr<strong>in</strong>. For our best knowledge this is the first report of goat h-<br />
lactoferr<strong>in</strong> transgenic pregnancies by us<strong>in</strong>g SCNT technique.<br />
P536<br />
Towards generation of human A20 gene transgenic pigs<br />
with improved features <strong>in</strong> xenotransplantation<br />
Oropeza, M 1,2 *, Petersen, B 1 , Herrmann, D 1 , Hassel, P 1 , Lucas-Hahn, A 1 ,<br />
Lemme, E 1 , Queisser, AL 1 , Niemann, H 1<br />
1Dept. of Biotechnology, Institute for Animal Breed<strong>in</strong>g, Mariensee, Neustadt,<br />
Germany; 2 University of Veter<strong>in</strong>ary Medic<strong>in</strong>e, Hannover, Germany<br />
Introduction Xenotransplantation is consi<strong>de</strong>red as promis<strong>in</strong>g to close<br />
the grow<strong>in</strong>g gap between <strong>de</strong>mand and availability of appropriate<br />
human organs. While the hyperacute rejection response can already be<br />
reliably controlled, the acute vascular rejection (AVR) rema<strong>in</strong>s a<br />
major hurdle for longterm survival of xenografts <strong>in</strong> a porc<strong>in</strong>e-toprimate<br />
organ transplantation. The human A20 (hA20) gene exhibits<br />
both antiapoptotic and anti-<strong>in</strong>flammatory properties <strong>in</strong> endothelial<br />
cells (Transplantation 82 : 36-40, 2006) and could thus prevent<br />
endothelial cell activation lead<strong>in</strong>g to AVR and xenograft <strong>de</strong>struction.<br />
The goal of this project is to produce transgenic pigs with improved<br />
features <strong>in</strong> xenotransplantation by transgenic expression of hA20.<br />
Materials and Methods The hA20-expression vector driven by the<br />
ubiquitous CAGGS hybrid promoter (chicken β-act<strong>in</strong>-/ rabbit β-<br />
glob<strong>in</strong>) conta<strong>in</strong><strong>in</strong>g an IRES-neomyc<strong>in</strong> resistance cassette (9.1 kb) was<br />
transfected <strong>in</strong>to porc<strong>in</strong>e fetal fibroblasts (PFFs) <strong>de</strong>rived from German<br />
Landrace porc<strong>in</strong>e fetal explant cultures. Transfection of 3 x 10 6 cells<br />
was accomplished at 450 V and 350µF with 10 µg plasmid DNA.<br />
G418-resistant (800 µg/mL) cell clones were screened by PCR with<br />
hA20 specific primers for hA20-<strong>in</strong>tegration. Somatic cell nuclear<br />
transfer (SCNT) was performed as recently <strong>de</strong>scribed (Clon<strong>in</strong>g Stem<br />
Cells 7: 35-44, 2005).<br />
Results A total of 80 cell clones were hA20-positive <strong>in</strong> PCR<br />
screen<strong>in</strong>g from 4 rounds of transfection after a 14 days G418 selection<br />
period. 30 positive cell clones were re-selected for 14 days with G418.<br />
Four of these cell clones were used as donor cells for SCNT.<br />
Immediately after SCNT, reconstructed embryos were transferred to a<br />
total of 10 synchronized recipient sows. Ultrasound exam<strong>in</strong>ation on<br />
day 23-25 of pregnancy confirmed established pregnancies <strong>in</strong> 7 of 10<br />
recipient sows. Two animals were sacrified on day 30 of pregnancy<br />
and a total of 8 fetuses were isolated. PCR and Southern Blot analysis<br />
showed <strong>in</strong>tegration of the hA20 gene <strong>in</strong> 6 of 8 fetuses.<br />
Conclusions The hA20-vector can be <strong>in</strong>tegrated <strong>in</strong> PFFs and hA20<br />
transgenic PFFs can successfully be used <strong>in</strong> SCNT to establish<br />
pregnancies. Further analysis will focus on the expression patterns <strong>in</strong><br />
A20-positive cell clones and the biological function of hA20 <strong>in</strong><br />
transgenic piglets. A second approach will be to <strong>in</strong>troduce an