Nuclear Transfer

ADVANCING CUTTING-EDGE TECHNOLOGY

NUCLEAR TRANSFER

BACKGROUND

Nuclear transfer (NT) is the technical term used for cloning. The technique was first developed with somatic cells in frogs in 1952 Briggs and King [1]. This was followed by Prof Sir John Gurdon’s discovery in 1966 that the nucleus of the Xenopus intestine cell can form every cell type in the body [2]. This crucial study revealed that the process of differentiation does not result in the permanent inactivation or loss of any genes in the genome, and that differentiated adult cells can be rejuvenated. Forty five years later, John Gurdon’s experimental design was applied by Ian Wilmut and Keith Campbell to mammals, producing Dolly the cloned sheep [3].

There are two types of somatic cell nuclear transfer (SCNT) – therapeutic cloning [4] and reproductive cloning [5], of which the former is not as strictly regulated. In therapeutic cloning, a patient can donate their somatic cells (e.g. skin cells) which are processed through a fine micropipette to isolate the differentiated nucleus and transfer it to an unfertilized egg whose nucleus has been removed [6]. After nuclear transfer, the egg with implanted nucleus is artificially activated and forms an embryo. Upon nuclear transfer, the host oocyte sets out to reprogram the transplanted somatic nucleus the way it would reprogram the sperm genome after fertilization [7].

Somatic Nuclear Transfer img

The cytoplasmic environment of the reconstructed zygote contains developmental components which reprogram the patient’s somatic nucleus into a state of totipotency, switching the differentiated cell nucleus back in time to its earliest embryonic state [8]. This happens via oocyte-specific components, such as linker histone B4 and histone H3.3, invading the transplanted nucleus, decondensing it and inducing expression of pluripotency genes within a few hours after NT [6, 9]. The newly expressed pluripotency genes are not expressed in adult cells. The inner cell mass of the produced embryo can be isolated to derive naïve pluripotent stem cells [10], a micromanipulation technique developed in 1997 [11]. Naïve pluripotent embryonic stem (ES) cells can grow in vitro indefinitely and can be used in differentiation protocols to generate any tissue of interest [4, 12]. Embryonic stem cells have been derived from cloned embryos of both primates and humans [10, 13]. Hence when a patient needs tissue replacement, SCNT and ES cell derivation can be used to generate a bank of tissues from their own genetic composition.

In 2006, the need for therapeutic embryonic cloning was alleviated by Takahashi and Yamanaka’s groundbreaking discovery that somatic cells can be reprogrammed to pluripotency by overexpressing specific transcription factors that are naturally expressed in ES cells [14]. The same effect was achieved – switching an adult cell back to the embryonic state – referred to as induced pluripotent stem cells (iPSC), and a new field of regenerative medicine began. Notably both SCNT and iPSC methods are highly inefficient, with success rates of 0.1 to 3% [9, 15], making this a major focus of research in regenerative medicine. Other than nuclear transfer to eggs or overexpression of transcription factors, somatic cell nuclear reprogramming can also be achieved by cell fusion, transdifferentiation, extract treatment, and transfer to immature oocytes [8, 16].

Research investigations in gametes of model organisms [17-24] and human gametes [13, 19, 25-28] have suggested that nuclear transfer techniques can be adapted for assisted reproductive technologies for treating certain fertility conditions. In this approach, instead of somatic cell nucleus, a nucleus from stage-matched egg or oocyte is transferred to a donor egg or oocyte for the healthy progression of embryonic development and generation of a healthy baby [22, 26]. In 2016, John Zhang - founder of Darwin Life - applied the nuclear transfer technique in assisted reproductive medicine to prevent the inheritance of a debilitating mitochondrial disorder of a patient in Mexico [26]. This resulted in a healthy birth from nuclear transfer – a groundbreaking demonstration of circumventing a severe genetically inherited disease [26]. Following this achievement, a similar nuclear transfer technique was applied to a 34-year old woman suffering from infertility and this resulted in another healthy birth from nuclear transfer in 2017, in Ukraine.