Andrea Lucas-Hahn

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.

Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development

The preimplantation bovine embryo is initially under the control of maternal genomic information that is accumulated during oogenesis. The genetic programme of development soon becomes dependent on new transcripts derived from activation of the embryonic genome. The early steps in development, including the timing of the first cleavage, activation of the embryonic genome, compaction and blastocyst formation, can be affected by the culture media and conditions, as well as the production procedure itself. These perturbations can possibly result in a marked decrease in the quality of the resulting blastocysts and may even affect the viability of offspring born after transfer. In vitro procedures such as in vitro production and somatic nuclear transfer of bovine embryos have been shown to be correlated with significant up- or downregulation, de novo induction or silencing of genes critical for undisturbed fetal and neonatal development. These alterations are likely to be caused by epigenetic modifications, such as DNA methylation and histone modifications. Analysis of perturbed epigenetic reprogramming and of the related phenomena, such as genomic imprinting and X-chromosome inactivation, in bovine embryos is promising for understanding the underlying mechanisms of developmental abnormalities, such as large offspring syndrome.

Gene Expression Patterns in Bovine In vitro-Produced and Nuclear Transfer-Derived Embryos and Their Implications for Early Development

Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.

Collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages

Ultrasound-guided follicular aspiration was performed on 29 Holstein-Friesian cows/heifers twice weekly at 3- to 4-d intervals over a period of 2 consecutive estrous cycles (total 42 d). For visualization of the ovaries and guidance of the aspiration needle, a 6.5 MHz fingertip probe on a 62 cm probe carrier was inserted into the vagina. The disposable aspiration needle was connected to a permanent rinse tubing system, thus ensuring minimum death of oocytes in the aspiration processs. After penetration of the vaginal wall, the needle was inserted into a follicle of the rectally fixed ovary. Cumulus oocyte complexes (COC) were aspirated at a pressure of 100 mm Hg. In the first experiment, the effect of an additional gonadotropin treatment 4 d prior to aspiration was investigated in 8 lactating cows. Following FSH-treatment, the number of aspirated follicles was higher (P < 0.05) than in the nontreated animals (10.6 +/- 0.7 vs 8.9 +/- 0.5). The number of recovered COC (7.0 +/- 0.6 vs 5.8 +/- 0.5), the recovery rate (COC per aspirated follicle) (66.6% vs 65.4%), the percentage of viable COC (56.8% vs 52.1%), the cleavage rate upon in vitro maturation and in vitro fertilization (56.7% vs 59.8%) as well as the rate of morula/blastocyst formation (3.8% vs 2.9%) were similar in both groups. In the second experiment, follicles were aspirated in 4 lactating cows, 6 dry cows, 4 pregnant cows (first 35 d of pregnancy), and 4 heifers. The average number of aspirated follicles and recovered COC was higher (P < 0.05) in the first 2 groups (10.6 +/- 0.6 and 9.3 +/- 0.7 follicles; 7.2 +/- 0.5 and 6.9 +/- 0.7 oocytes) than in trie 2 other treatment groups (7.3 +/- 0.5 and 8.1 +/- 0.5 follicles; 5.0 +/- 0.4 and 5.7 +/- 0.5 oocytes). The percentage of viable COC was higher (P < 0.05; 68.3%) in lactating animals than in all the other groups (49.7, 52.5 and 57.4%, respectively). Similarly, upon in vitro fertilization, cleavage rate was higher (P < 0.05; 63.4%) in lactating cows than in the other groups (43.7, 50.5, 55.1%, respectively). A total of 21.5, 22.7, 11.9 and 13.5%, respectively, in the 4 groups of the in vitro fertilized oocytes reached the morula and blastocyst stages. After transfer of a total of 48 embryos 22 pregnancies (45.8%) were established as detected on Day 65. We conclude that 1) repeated aspiration of viable COC at short intervals is possible, 2) additional FSH-treatment does not increase oocyte yields, and 3) viable blastocysts can be produced from cattle at various reproductive phases irrespective of the reproductive phase.

Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.

Oropeza M, Petersen B, Carnwath JW, Lucas‐Hahn A, Lemme E, Hassel P, Herrmann D, Barg‐Kues B, Holler S, Queisser A‐L, Schwinzer R, Hinkel R, Kupatt C, Niemann H. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.
Xenotransplantation 2009; 16: 522–534.

Pigs transgenic for human thrombomodulin have elevated production of activated protein C.

Petersen B, Ramackers W, Tiede A, Lucas‐Hahn A, Herrmann D, Barg‐Kues B, Schuettler W, Friedrich L, Schwinzer R, Winkler M, Niemann H. Pigs transgenic for human thrombomodulin have elevated production of activated protein C.
Xenotransplantation 2009; 16: 486–495.

Development and Validation of a Highly Efficient Protocol of Porcine Somatic Cloning Using Preovulatory Embryo Transfer in Peripubertal Gilts

The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50% and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production.

DNA Methylation Patterns Reflect Epigenetic Reprogramming in Bovine Embryos

To understand the epigenetic alterations associated with assisted reproduction technology (ART) and the reprogramming of gene expression that follows somatic cell nuclear transfer (SCNT), we screened a panel of 41 amplicons representing 25 developmentally important genes on 15 different chromosomes (a total of 1079 CpG sites). Methylation analysis was performed on DNA from pools of 80 blastocysts representing three classes of embryos. This revealed a subset of amplicons that distinguish between embryos developing in vivo, produced in vitro, or reconstructed by SCNT. Following SCNT, we observed massive epigenetic reprogramming evidenced by reduced levels of methylation in the resultant embryos. Analysis of data from the 28 most informative amplicons (hotspot loci), representing more than 523 individual CpG sites, we discovered subsets of amplicons with methylation patterns that were unique to each class of embryo and may indicate metastable epialleles. Analysis of eight genes with respect to mRNA expression did not reveal a direct correlation with DNA methylation levels. In conclusion, this approach revealed a subset of amplicons that can be used to evaluate blastocyst quality and reprogramming following SCNT, and can also be employed for the localization of the epigenetic control regions within individual genes and for more general studies of stem cell differentiation.