Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change.

BACKGROUND: High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing. RESULTS: Using gRNA/Cas9-mediated editing, we introduced a three bp deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as causative variant for the semi-dominant color dilution phenotype observed in Galloway and Highland cattle. Calves generated from cells with homozygous edits revealed a strong color dilution effect. Instead of the characteristic black and white markings of control calves generated from unedited cells, the edited calves displayed a novel grey and white coat pattern. CONCLUSION: This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. Although only one of the calves was healthy at birth and later succumbed to a naval infection, the study showed the feasibility of generating such edited animals with the possibility to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change.

Keith's MAGIC: Cloning and the Cell Cycle.

Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.

Cattle cloned from increasingly differentiated muscle cells.

It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1-6 days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH, and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92%, 85%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 for MPC vs. MT vs. MF, respectively, P < 0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively).

Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages.

Expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated and nuclear transfer derived bovine pregnancies.

Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

Animal cloning: problems and prospects.

An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer acceptance. Future improvements in animal cloning will largely arise from a greater understanding of the molecular mechanisms of reprogramming.

Couplet alignment and improved electrofusion by dielectrophoresis for a zona-free high-throughput cloned embryo production system.

Mammalian cloning by somatic nuclear transfer has great potential for developing medical applications such as biopharmaceuticals and generation of tissues for transplantation. For agricultural applications, it allows the rapid dissemination of genetic gain in livestock breeding. The maximisation of that potential requires improvements to overall cloning technology, especially with respect to increasing cloning efficiency and throughput rates in cloned embryo production. A zona-free embryo reconstruction system was developed to increase cloning throughput and ease of operation. Central to this system is a modified electrofusion procedure for nuclear transfer. Cytoplast-donor cell couplets were placed in a custom-designed 'parallel plate' electrode chamber. A 1 MHz sinusoidal AC dielectrophoresis alignment electric field of 6-10 kV m(-1) was applied for 5-10s. The couplets were then fused using 2 x 10 micros rectangular DC-field pulses (150-200 kV m(-1)), followed by application of the AC field (6-10 kV m(-1)) for another 5-10 s. Fusion was performed in hypoosmolar buffer (210 mOsm). Automated alignment of up to 20 couplets at a time has been achieved, resulting in greatly improved fusion throughput rates (2.5-fold increase) and improved fusion yields (1.3-fold increase), compared with commonly followed zona-intact protocols.

The health of somatic cell cloned cattle and their offspring.

The cloning syndrome is a continuum with the consequences of abnormal reprogramming manifest throughout gestation, the neo-natal period, and into adulthood in the cloned generation, but it does not appear to be transmitted to subsequent offspring following sexual reproduction. Most in vivo studies on bovine somatic cell cloning have focused on development during pregnancy and the neo-natal period. In this paper, we report on the viability and health of cloned cattle in adulthood. From our studies at AgResearch, we find that between weaning and 4 years of age, the annual mortality rate in cattle cloned from somatic cells is at least 8%. Although the reasons for death are variable and some potentially preventable, the main mortality factor in this period is euthanasia due to musculoskeletal abnormalities. This includes animals with severely contracted flexor tendons and those displaying chronic lameness, particularly in milking cows. In contrast, no deaths beyond weaning have so far been encountered with the offspring of clones where the oldest animals are 3 years of age. In surviving cloned cattle, blood profiles and other indicators of general physiological function such as growth rate, reproduction, rearing of offspring, and milk production are all within the normal phenotypic ranges.

Cloned cattle derived from a novel zona-free embryo reconstruction system.

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.

Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle.

Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G(0), G(1), and different phases within G(1). We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G(0) results in a significantly higher percentage of viable calves at term than synchronization in early G(1) or late G(1). For transgenic fibroblasts, however, cells selected in G(1) show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G(0). This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.

Cloning cattle.

Over the past six years, hundreds of apparently normal calves have been cloned worldwide from bovine somatic donor cells. However, these surviving animals represent less than 5% of all cloned embryos transferred into recipient cows. Most of the remaining 95% die at various stages of development from a predictable pattern of placental and fetal abnormalities, collectively referred to as the "cloning-syndrome." The low efficiency seriously limits commercial applicability and ethical acceptance of somatic cloning and enforces the development of improved cloning methods. In this paper, we describe our current standard operating procedure (SOP) for cattle cloning using zona-free nuclear transfer. Following this SOP, the output of viable and healthy calves at weaning is about 9% of embryos transferred. Better standardization of cloning protocols across and within research groups is needed to separate technical from biological factors underlying low cloning efficiency.

Somatic cell nuclear transfer.

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.

Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle.

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.

Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells.

Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.

Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed.

To preserve the female genetics of an endangered breed of cattle, adapted to sub-Antarctic conditions, adult somatic cell nuclear transfer was used to clone the last surviving Enderby Island cow from mural granulosa cells. Embryos reconstructed with metaphase II cytoplasts and quiescent cells were either activated and fused simultaneously (AFS) at 24 or 30 hours post maturation (hpm) or alternatively, fused 4-6 h before activation at 26-30 hpm (FBA). A significantly higher proportion of fused embryos developed in vitro to grade 1-3 blastocysts on Day 7 with FBA (39.8+/-2.8%) compared to AFS with activation either at 24 hpm (10.6+/-3.9%, P<0.01) or at 30 hpm (18.6+/-4.1%, P<0.01). Following the transfer of 74 embryos from the FBA treatment over two experiments, survival rates on Days 30, 55, 85, 150 and 190 of pregnancy were 38%, 30%, 23%, 16% and 15%, respectively. Of 22 embryos transferred in the first experiment, two calves were born alive with one calf surviving. DNA analyses confirmed that the calves were genetically identical to the Enderby Island cow. Additional pregnancies are currently ongoing. These data show that embryo development is increased by prolonged exposure of quiescent somatic cell nuclei to oocyte cytoplasm before artificial activation, possibly facilitating nuclear reprogramming. The successful demonstration of somatic cell nuclear transfer in animal conservation extends the applications of the technology beyond the main agricultural and biomedical interests.

Cloning sheep from cultured embryonic cells.

The production of transgenic farm animals will be greatly enhanced with the development of cultured cell lines that remain totipotent following nuclear transfer. Here, data are presented that demonstrate the generation of both male and female cloned lambs from two established embryonic cell lines. Cytoplasts derived from in vivo oocytes resulted in slightly greater development to blastocyst (24% v. 17%) and survival to term (7% v. 2%) compared with in vitro oocytes. There was no advantage in co-culturing cloned embryos with oviductal epithelial cells compared with synthetic oviductal fluid medium in terms of development to blastocyst (18% v. 31%) or survival to term (both 8%). Although the survival of cloned embryos immediately after transfer was high based on 'biochemical' pregnancy, 64-80% of embryos failed over the attachment phase with in vivo cytoplasts. Although the co-transfer of trophoblastic vesicles improved embryo survival to Day 35 (45% v. 25%), there was no difference at term. A high proportion of fetuses were lost during the last trimester (43%), resulting in 11% of embryos transferred developing to term using in vivo cytoplasts (12/112). Five lambs have survived and two rams are fertile. The current nuclear transfer process is inefficient and further research is needed to improve the development of healthy fetuses.

Production of cloned lambs from an established embryonic cell line: a comparison between in vivo- and in vitro-matured cytoplasts.

Nuclear transfer procedures were used to determine the in vivo developmental potential of an ovine embryonic cell line isolated from the inner cell mass of a Day 8 blastocyst-stage embryo. This cell line possessed a differentiated epithelial-like cell morphology. In this study, a comparison was made between in vivo- and in vitro-derived oocytes used as recipient cytoplasts in the nuclear transfer procedure. Cultured cells were induced to quiesce and enter presumptive G0 before being used as donor karyoplasts between passages 8 and 16 of culture. After cell fusion, reconstructed embryos were cultured for 6 days in vitro in embryo culture medium. Blastocyst-stage embryos were subsequently transferred to synchronized recipient ewes (n = 37), and development was allowed to proceed to term. There was a significant effect of source of recipient cytoplast, with development being consistently greater with in vivo compared to in vitro cytoplasts in terms of, respectively, blastocysts produced (24.2 +/- 3.8% vs. 17.1 +/- 2.3%; p = 0.1), Day 35 pregnancy rate (40.0% vs. 9.1 %; p < 0.05), and Day 35 embryo survival (19.4% vs. 4.5%; p < 0.05). A high proportion of fetuses died during late gestation (5 of 8). The major abnormalities were associated with the urogenital tract. However, three lambs were delivered alive following cesarean section on Day 147. One lamb, derived from an in vitro-matured oocyte, died after 10 min, while the remaining two from in vivo-ovulated oocytes are apparently normal and healthy. DNA microsatellite markers conclusively show that the three lambs are genetically identical and were derived from the embryonic cell line. In conclusion, some cells from this blastocyst-derived embryonic cell line are totipotent by nuclear transfer and can produce viable offspring.

ES cell cycle rates affect gene targeting frequencies.

We have investigated the gene targeting frequency at the hprt locus in a range of embryonic stem cell lines selected for variations in cell cycle parameters. Our results show that targeting frequency varies with cell line by as much as 12-fold between nonisogenic lines and 3-fold between isogenic lines and that a nonisogenic line can support homologous recombination events by up to 21-fold more frequently than an isogenic line. This variation is consistent with both insertion and replacement vectors. These results can be explained by an inverse linear correlation of targeting frequencies with cell doubling times. Additionally, by reducing serum concentration in the culture medium the mean cell doubling time for R1 ES cells can be increased from 11.4 to 15.7 h, with a subsequent 15-fold decrease in gene targeting frequency. This change fits the correlation found for the different nonisogenic cell lines. Our observations have important implications when performing gene targeting experiments and explain some of the variation noted between experiments.