46 RECLONING USING TRANSGENIC FETAL FIBROBLASTS AS NUCLEI DONORS INCREASES DEVELOPMENTAL POTENTIAL OF RECONSTRUCTED EMBRYOS IN CATTLE

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P > 0.05) between control and recloned groups regarding fusion rate (n = 550, 62.22% and n = 189, 57.25%; respectively) or cleavage rate (n = 383, 69.45% and n = 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P < 0.01) compared with the control group (n = 51, 26.98%, and n = 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n = 6, 15.38% and n = 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil.

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Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽

10.1530/rep.1.01206 ◽

2007 ◽

Vol 133 (1) ◽

pp. 219-230 ◽

Cited By ~ 73

Author(s):

Feikun Yang ◽

Ru Hao ◽

Barbara Kessler ◽

Gottfried Brem ◽

Eckhard Wolf ◽

...

Keyword(s):

Histone Acetylation ◽

Nuclear Transfer ◽

Donor Cell ◽

Epigenetic Mark ◽

Cell Type ◽

Developmental Potential ◽

Acetylation Status ◽

Donor Cells ◽

Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

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Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Development ◽

10.1242/dev.108.1.185 ◽

1990 ◽

Vol 108 (1) ◽

pp. 185-189 ◽

Cited By ~ 4

Author(s):

J.N. Petitte ◽

M.E. Clark ◽

G. Liu ◽

A.M. Verrinder Gibbins ◽

R.J. Etches

Keyword(s):

Inbred Line ◽

Cell Lineage ◽

Donor Cell ◽

Black Pigment ◽

Dominant Allele ◽

White Leghorn ◽

Sperm Dna ◽

Donor Cells ◽

White Leghorns ◽

Blastodermal Cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)

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102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab102 ◽

2007 ◽

Vol 19 (1) ◽

pp. 168

Author(s):

V. Zakhartchenko ◽

F. Yang ◽

R. Hao ◽

E. Wolf

Keyword(s):

Histone Acetylation ◽

Nuclear Transfer ◽

Epigenetic Mark ◽

Cloning Efficiency ◽

Developmental Potential ◽

Acetylation Status ◽

Donor Cells ◽

Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P < 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P < 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P < 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P < 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

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29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab29 ◽

2012 ◽

Vol 24 (1) ◽

pp. 126

Author(s):

X. Yang ◽

J. Mao ◽

E. M. Walters ◽

M. T. Zhao ◽

K. Lee ◽

...

Keyword(s):

Somatic Cell ◽

Embryo Development ◽

Natural Science ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Control Group ◽

Blastocyst Formation ◽

Donor Cells ◽

Fetal Fibroblasts ◽

Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

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357 EARLY FETAL DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS GENERATED FROM FIBROBLASTS GENETICALLY MODIFIED BY piggyBac TRANSPOSITION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab357 ◽

2015 ◽

Vol 27 (1) ◽

pp. 266

Author(s):

A. Alessio ◽

A. Fili ◽

D. Forcato ◽

M. F. Olmos-Nicotra ◽

F. Alustiza ◽

...

Keyword(s):

Fetal Development ◽

Nuclear Transfer ◽

Fluorescent Protein ◽

Genetically Modified ◽

Bovine Genome ◽

Control Group ◽

Large Animal ◽

Phenotypic Analysis ◽

Fluorescence Excitation ◽

Fetal Fibroblasts

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117–8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19 184–19 189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey's test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0 ± 17.8 v. 100.0 ± 16.1 and 2.8 ± 0.8 respectively, n = 4–7; P < 0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development. Table 1.SCNT and early fetal development of bovine fibroblasts transposed with piggyBac1 The financial support of UNRC, CONICET and ANPCyT from Argentina is gratefully acknowledged.

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Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

Zygote ◽

10.1017/s096719941000064x ◽

2011 ◽

Vol 20 (1) ◽

pp. 61-66 ◽

Cited By ~ 13

Author(s):

Ying Liu ◽

Olga Østrup ◽

Juan Li ◽

Gábor Vajta ◽

Lin Lin ◽

...

Keyword(s):

Cell Membranes ◽

Time Window ◽

Donor Cell ◽

Egg Extract ◽

Donor Cells ◽

Fetal Fibroblasts ◽

Blastocyst Rate ◽

Pre Treatment ◽

Adult Fibroblasts

SummaryPre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.

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72 EFFECT OF CELL CYCLE PHASE OF GENE-MANIPULATED FETAL FIBROBLASTS ON THE DEVELOPMENT OF CLONED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab72 ◽

2005 ◽

Vol 17 (2) ◽

pp. 186

Author(s):

M. Urakawa ◽

T. Sawada ◽

Y. Sendai ◽

Y. Shinkai ◽

A. Ideta ◽

...

Keyword(s):

Cell Cycle ◽

Donor Cell ◽

Developmental Rate ◽

G1 Phase ◽

Control Group ◽

Rate Of Development ◽

Normal Fetus ◽

Fetal Fibroblasts ◽

Blastocyst Rate ◽

And Control

Transgenic bovine fetuses and offspring can be produced by using gene-modified somatic cells and clones of these cells. In this study, we examined the effects of specific cell cycle (early G1 phase) of donor cell (gene-manipulated fibroblasts) on the development of the nuclear transfer (NT) embryos into blastocysts and on the fetus production after embryo transfer. The gene-manipulated (tg; targeting of one or both alleles of gene encoding α-1,3-galactosyltransferase) or non-manipulated (control) bovine fetal fibroblasts were used for NT. The fibroblasts transfected with the targeting vector were selected with 0.4 mg mL−1 G418. The G418-resistant cells were monitored by PCR and Southern blot analysis. The cells (tg cells) in which homologous recombination occurred were used for NT. For NT, both tg cells and control cells were cultured in DMEM with 10% FCS. Early G1 cells were prepared by choosing pairs of bridged cells derived from mitotic phase cells (Urakawa M et al. 2004 Theriogenology 62, 714–728), and non-synchronized cells were obtained from a culture plate that had reached 60–80% confluence. Each donor cell was inserted into an enucleated, in vitro-matured (19 h) oocyte. Oocyte-cell couples were electrofused and activated with calcium ionophore and cycloheximide. The NT embryos were then co-cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were determined at 6 days after NT. The blastocysts were nonsurgically transferred to recipient heifers, and the developmental rate to the normal fetus was examined by the recovery of fetus or by using ultrasonography at Days 35–42. Data were analyzed by ANOVA. The developmental rate to the blastocyst stage did not differ significantly between tg (28.4%, 128/425) and control (25.4%, 181/739) cell groups. In the control group, the blastocyst rate of embryos constructed from early G1 phase fibroblasts (25.7%, 80/311) was not significantly different from that of embryos constructed with non-synchronized fibroblasts (23.6%, 101/428). In contrast, the blastocyst rate of tg cell derived-embryos was lower (P < 0.05) in early G1 phase (23.5%, 71/302) than in non-synchronized cell phase (46.3%, 57/123). The rate of development to a normal fetus in the tg group (15.4%, 4/26) was significant lower than that in the control group (62.5%, 25/40). For both the tg group and the control group, the rate of development to fetus tended to be higher (P > 0.05) for blastocysts derived from cells at the early G1 phase than for blastocysts derived from non-synchronized cells (tg group, 25.0%, 3/12 v. 7.1%, 1/14; control group, 90.0%, 9/10 v. 53.3%, 16/30). These results demonstrate that gene modification of fetal fibroblasts affects the development of NT embryos to fetuses. In addition, the synchronization of genetically modified donor cells to the early G1 phase may increase the potential to develop to a normal fetus.

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Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

10.32469/10355/66732 ◽

2017 ◽

Author(s):

◽

Bethany Rae Mordhorst

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Nuclear Reprogramming ◽

In Vitro Development ◽

Fetal Fibroblasts ◽

Pharmaceutical Agents ◽

Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

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309 PRODUCTION OF CATTLE BY NUCLEAR TRANSFER FROM CELLS IN WHICH A GENE IS DISRUPTED

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab309 ◽

2009 ◽

Vol 21 (1) ◽

pp. 251

Author(s):

M. Urakawa ◽

Y. Sendai ◽

A. Ideta ◽

K. Hayama ◽

Y. Shinkai ◽

...

Keyword(s):

Pregnancy Rate ◽

Gene Targeting ◽

Prion Protein ◽

Nuclear Transfer ◽

Antibiotic Resistance Gene ◽

Control Group ◽

Gestation Length ◽

Entry Site ◽

Donor Cells ◽

Fetal Fibroblasts

Gene-targeted animals provide a powerful model to examine gene functionality. In this study, we examined the effect of gene targeting of donor cells for nuclear transfer (NT) on the pregnancy rate and on viability of the offspring after embryo transfer. Gene-targeted (tg; targeting of both alleles of the gene encoding bovine prion protein) or non-manipulated (control) bovine fetal fibroblasts were used for NT. A promoterless positive selection vector (pPrP5.2) containing an internal ribosome entry site-antibiotic resistance gene (neo) cassette and loxP sequences was used to disrupt the bovine prion protein gene. The cells (tg) in which homologous recombination was occurred were used for NT. The tg and control cells were cultured in DMEM with 10% FCS and were prepared in the early G1 phase to our previous report (Urakawa M et al. 2004 Theriogenology 62, 714–728). Each donor cell was inserted into an enucleated in vitro-matured (19 h) oocyte. Cell fusion (DC, 200 V mm–1, 10 μs) and activation (DC, 100 V mm–1 , 60 μs) were done in 0.3 m mannitol solution. The NT embryos were then activated with 5 μm Ca-ionophore and 10 μg of mL–1 cycloheximide and were cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were judged at 6 days after NT. The blastocysts were non-surgically transferred to recipient heifers. The recipients were monitored daily for heat behavior, examined by ultrasound at Day 30 and 60, and then observed monthly to confirm pregnancy. The offspring born in the tg group were confirmed by PCR to be transgenic. Statically significance was tested using a chi-square test or t-test. Developmental rate to the blastocyst stage, pregnancy rate at Day 30 and 60, and calving rate did not differ significantly between tg and the control group (Table 1). Gestation length (tg; 290.0 ± 2.2 days v. control; 290.5 ± 3.9 days) and birth weight (tg; 39.6 ± 8.0 kg v. control; 40.2 ± 4.1 kg) were not significantly different. These results indicate that gene targeting of donor cells used for NT does not significantly affect the development of embryos, pregnancy rate, or the viability of the offspring. Table 1.Development of NT embryos with tg or control cells

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259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab259 ◽

2007 ◽

Vol 19 (1) ◽

pp. 246

Author(s):

B. Mohana Kumar ◽

H. F. Jin ◽

J. G. Kim ◽

S. A. Ock ◽

H. J. Song ◽

...

Keyword(s):

Dna Methylation ◽

Expression Pattern ◽

Nuclear Transfer ◽

Total Cell ◽

Dna Hypomethylation ◽

Developmental Potential ◽

Expression Of Genes ◽

Donor Cells ◽

Fetal Fibroblasts

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P < 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P < 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P < 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

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