Generation of Genetically Engineered Livestock Using Somatic Cell Nuclear Transfer

Abstract

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985 – 1996). The applications of technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of Somatic Cell Nuclear Transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996 – 2013), SCNT was the only method used for generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

Telomere Dynamics in Livestock

“…The use of somatic cell nuclear transfer (SCNT) in the livestock industry has provided the opportunity to clone genetically superior animals and produce cloned transgenic animals [85]. One concern regarding SCNT is when using an aged somatic cell for cloning the offspring might start life with shortened telomeres, resulting in earlier cell senescence.…”

Modificación genética en cerdos destinados a xenotrasplante

“…La combinación de las metodologías de modificación genética con la transferencia nuclear es fundamental para la obtención de cerdos adecuados para xenotrasplante. La transferencia nuclear de células somáticas (SCNT, somatic cell nuclear transfer) implica transferir el núcleo de una célula somática a un oocito enucleado, produciéndose la reprogramación epigenética de la célula donante y generándose un nuevo embrión, que es transferido a una hembra receptora pseudogestante en la que tendrá lugar la gestación (Polejaeva, 2021). Los fibroblastos fetales porcinos son las células más utilizadas como donantes de núcleo (Ouyang et al, 2021); estas células se pueden modificar genéticamente en cultivo para, posteriormente, generar animales modificados genéticamente mediante SCNT.…”

Dolly at 25… is she ‘… still goin’ strong?’

“…There then follows two articles that each provide both a historical and contemporary perspective on the role of SCNT in the generation of GM livestock. The first article ( Polejaeva 2021 ) provides a detailed overview of the technical advances made in GM technologies over the past 25 years from a leading member of the team that produced the first cloned pigs by SCNT in 2000. This article also offers a perspective on the current and future role of SCNT in the creation of genetically engineered animals; a perspective that is extended by Galli & Lazzari (2021) who consider applications in several large-animal species together with intellectual-property and regulatory challenges that will ultimately determine its use.…”