Cloned Cattle Derived from a Novel Zona-Free Embryo Reconstruction System

Abstract

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.

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

“…Concerns about removing zona from embryos include the possibility of viral infection of them (however the risk is the same for conventional cloning, due to the rupture of the zona), the potential failures in DNA methylation in these embryos, as shown for mouse zona free embryos [18] and the impossibility for group culture of embryos due to aggregation of adjacent embryos; however this problem has been overcame by the use of WOW culture [3]. In the last years this method has been successfully used for production of bovine [2,[19][20][21], horse [22][23], mouse [24] and pig [25] offspring. Finally, and probable one of the most important features of HMC is its simplicity, speed of work and low cost when compared to traditional cloning using micromanipulators.…”

“…Attempts to simplify cloning had been made [1][2][3][4] the main focus being to avoid the use of expensive equipment, such as micromanipulators, inverted microscopes and pulling and bevelling devices for microinstruments. All these changes are included in the so called hand made cloning (HMC) approach developed originally in 2001 [5][6][7].…”

Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

“…Nuclear transfer was performed using a zona-free method as described (Oback et al 2003, Lagutina et al 2007). In brief, in vitro matured oocytes were denuded of cumulus cells by gentle vortexing and /or pipetting after brief exposure to hyaluronidase (300 IU/ml), and oocytes with a visible first polar body were selected for nuclear/spindle transfer or parthenogenetic activation.…”

Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

“…Generation of IVP embryos by in vitro fertilisation (using sperm from bull AESF 1) is as previously described (Thompson et al 2000) except for zona removal after fertilisation and single embryo culture. Bovine NT, using cultured skin fibroblasts recovered from bull AESF 1 and embryo culturing of both the IVP and NT embryos using a synthetic oviduct fluid system, with embryos cultured singly was performed as described elsewhere (Oback et al 2003). Grading and staging of development according to published guidelines (Robertson & Nelson 1998) was performed by only one of us (D N W).…”