Nuclear Transfer in Cloning | Animal Biotechnology

In this article we will discuss about the process of nuclear transfer in cloning of animals.

Current Efficiency of Somatic Cell Nuclear Transfer:

Overall, the current efficiency of NT with somatic cells is poor. At AgResearch, the proportion of reconstructed 1 -cell cattle embryos that develop to transferable quality blastocysts after seven days of culture (40%) is comparable to that following in vitro embryo production (IVP) (i.e. in vitro matured, fertilised and cultured) with abattoir- derived oocytes.

However, at present, in vivo development is only one- third that following IVP. For example, of 988 somatic cell cloned embryos transferred at AgResearch, only 13% resulted in calves delivered at full term. This compares with 30% to 45% embryo survival with IVP.

Although pregnancy rates in cattle on day 50 of gestation after the transfer of single NT embryos can be as high as 65%, and similar to both IVP embryos and artificial insemination, there is continual loss thereafter with the clones.

Moreover, perinatal and post-natal mortality rates with cloned offspring are greater than normally expected, with only 64% of cloned calves surviving to weaning at three months of age. Recently, the concerns regarding the long-term health and survival of clones into adulthood have been more fully appreciated.

From an animal welfare perspective, in addition to farmer and consumer acceptance of the technology, these losses must be solved before any large-scale cloning opportunities are practicable or tolerated.

Ideally, cloning efficiency should have pregnancy rates comparable to those achieved following sexual reproduction, principally artificial insemination or after transfer of in vivo produced embryos, which is 55% to 60%.

It is important to remember, however, that somatic cell NT can be effective in producing what appear to be physiologically normal animals. This provides encouragement for eventually resolving technical issues and elucidating the molecular mechanisms responsible for effecting complete epigenetic reprogramming.

Complete Reprogramming:

There is evidence that some cloned animals are physiologically normal or at least they may develop a stable metabolism sometime after birth. It is remarkable that somatic cell NT is successful, for a tremendous amount is asked of a differentiated donor nucleus to re-establish the correct pattern of gene expression to allow normal embryogenesis.

Various international studies in a range of species provide evidence that some clones appear to be the same as their non-clone counterparts in the following areas:

i. Behaviour

ii. Growth rates

iii. Reproduction

iv. Livestock production characteristics

v. Life spans.

Furthermore, the sexually-derived offspring of clones also appear normal.

Incomplete Reprogramming:

There are instances, however, where reprogramming appears to be incomplete. For normal development following NT, it is generally accepted that the epigenetic modifications in the donor nucleus must be reprogrammed to a state comparable to those in a zygote.

This is necessary for the correct pattern of gene expression to occur during subsequent embryogenesis. This reprogramming must occur within a short timeframe, in a different cellular context compared with normal development, and is prone to error.

There are increasing amounts of data documenting deviations in epigenetic reprogramming, with clones showing inappropriate patterns of following:

i. DNA metnylation

ii. Chromatin modification

iii. X-chromosome inactivation

iv. Expression of imprinted and non-imprinted genes.

The pattern of mortality and clone phenotypes observed presumably reflect the inappropriate expression of various genes, whose harmful effects are exerted at various different stages of development. Aberrations that occurred early in embryonic or foetal development may impair health in adulthood.

There is a wide spectrum of phenotypic outcomes, ranging from those that are lethal to those that appear neutral. Outcomes which appear neutral do not compromise the health and welfare of the animal, but the epigenetic variations reduce the uniformity in the clonal family, which may be undesirable for some applications.

The common consequences of incomplete reprogramming following somatic cell NT are:

i. Higher rates of pregnancy loss

ii. Difficult parturition

iii. Higher rates of post-natal mortality

iv. Some specific clone-associated phenotypes in adulthood.

Placental Abnormalities:

A failure of the placenta to develop and function correctly is a common feature amongst clones. The majority of early pregnancy failures, before placentome formation, are attributed to an inadequate transition from yolk sac to allantoic-derived nutrition, with poor allantoic vascularisation in sheep.

Furthermore, there is reported evidence of immunological rejection contributing to early embryonic loss. Typically in cattle, 50% to 70% of pregnancies at day 50 are lost throughout the remainder of gestation and up to term. This is in stark contrast to only 0% to 5% loss with artificial insemination or natural mating over the same period.

In extreme cases, placentomes are entirely absent at day 50. Shortly thereafter, these pregnancies fail. More commonly, cloned placentae only have half the normal number of placentomes, display compensatory overgrowth and are oedematous.

Of particular concern are the losses in the second half of gestation: especially the occurrence of hydroallantois, i.e. the excess accumulation of fluid within the allantois. Hydroallantois does occur with other forms of assisted sexual reproduction in cattle, ranging in incidence between 0.07% to 5% for artificial insemination and IVP, respectively.

With clones, however, typically 25% of those cows pregnant at day 120 of gestation develop clinical hydroallantois (hydrops). The range is variable (0% to 40%) with the incidence possibly dependent upon the individual cell line.

Cases are typically severe enough to render the calves nonviable and on welfare grounds, in order to reduce the risk of mortality to the recipient cow, the standard practice is to electively terminate the hydrops pregnancy. This is an unsatisfactory management procedure and work is underway to identify non-viable pregnancies much earlier in development to lessen the welfare burden.

Ideally, a reprogramming marker would enable only viable embryos to be transferred into recipients. Alternatively, abnormal pregnancy development could be determined by measuring specific components present in maternal serum with early detection allowing early elective abortion.

The level of pregnancy specific protein-b produced by the binucleate cells of the trophoblast was transiently higher at day 35 in those concept that failed to develop today 90. Similarly, levels of pregnancy serum protein 60 were elevated over the first four months of gestation in those pregnancies that became pathological. Pregnancy monitoring is complemented by detailed ultrasonography.

Parturition Difficulties:

Intervention is often deemed necessary to deliver cloned offspring, as gestation length in NT pregnancies is typically prolonged and the birth weight of cloned calves may be 25% heavier than normal.

Newborn cloned calves display functional adrenal glands, so this extended gestation may be due to failure of the placentae to respond to foetal Cortisol near term or to a lack of adrenocorticotropic hormone release from the foetus. Oversized cloned offspring add to the birth complications. They are larger than IVP, artificially inseminated or naturally-mated controls. It has been reported that somatic cloned calves are heavier than embryonic clones.

At AgResearch, the occurrence of prolonged gestation and the risk of dystocia initially prompted the delivery of clones by elective caesarean-section, following a brief exposure to exogenous corticosteroids. Recognising the welfare issues and the intensive peri­natal veterinary care often required, we have modified our calving management system.

The aim is to have a planned vaginal delivery (with manual traction if necessary), using an alternative corticosteroid therapy to aid foetal maturation, especially of the lungs, and to completely induce parturition a week before expected full term.

This protocol has reduced the incidence of caesarean-section to 5% to 10% and with the majority of cloned calves reared on their recipient dams. Although not completely natural, this approach towards delivering cloned calves in a controlled manner is feasible and acceptable on farm.

Post-Natal Viability:

The viability of cloned offspring at delivery and up to weaning is reduced compared to normal, and this is despite greater than usual veterinary care. Data from our group shows that around 80% of cloned calves delivered at term are alive after 24 hr.

Two-thirds of the mortality within this period is due to a spinal fracture syndrome through the cranial epiphyseal plate of the first lumbar vertebrae or to deaths that occurred either in utero or from dystocia. Surviving newborn clones have altered neonatal metabolism and physiology, possibly due to placental abnormalities, and it takes time for these processes to adjust to normal.

At AgResearch, typically an additional 15% of calves initially born alive die before weaning. In our experience, the most common mortality factors during this period are gastroenteritis and umbilical infections.

Other abnormalities noted include defects in the cardiovascular, musculoskeletal and neurological systems, as well as susceptibility to lung infections and digestive disorders. Hydronephrosis is particularly common in sheep, with correspondingly elevated serum urea levels in some surviving clones.

The proportion of cloned calves born that are longer-term survivors ranges between 47% and 80%. AgResearch data show that the stage of the donor cell cycle at the time of NT affects subsequent calf viability.

The proportion of cloned calves that survive to weaning is significantly greater for those derived from quiescent GO donor cells (81 %) than for those derived from G1 cells (50%). Post-natal survival of cloned sheep is substantially less than that of cattle with both somatic and embryonic cell types (31 % and 42%, respectively).

Clone-Specific Phenotypes:

Whilst there are some studies indicating that clones can be physiologically normal and apparently healthy, there are other observations and reports in the literature of abnormal clone-associated phenotypes that become apparent during the juvenile and adult phases of life.

The incidence of these anomalies may vary according to the particular species, genotype or cell type, or according to specific aspects of the NT and culture protocols used.

More research is required to determine the following:

i. The effects of nuclear-mitochondrial interactions arising from a donor nucleus in a foreign cytoplasmic environment

ii. The effects of mitochondrial DNA heteroplasmy and possible recombination events on cloning efficiency – the resulting fitness of the cloned animals.

The cloned offspring syndrome is a continuum, in that lethality or abnormal phenotypes may occur at any phase of development, depending upon the degree of dysregulation of key genes, presumably due to fundamental errors in epigenetic reprogramming. Even apparently normal clones may have abnormal regulation of many genes that are too subtle to result in an obvious phenotype.

Telomeres are regions of DNA at the ends of chromosomes which progressively shorten after each cell division in most somatic cell types. Whilst Dolly may have developed arthritis and was euthanised at a relatively young age because of a virally induced lung tumour, this may have resulted from her largely indoor housing and handling rather than the fact that she was a clone. Other studies have been contradictory with regard to telomere length in clones, with reports of restoration to normal in cattle and mice and even instances of extended telomere lengths.

The discovery of a telomere length restoration process that occurs during early embryogenesis appears responsible for this. Normal telomere lengths have even been reported after repeated re-cloning in mice and cattle and specifically, in the spermatozoa of somatic cell cloned bulls and subsequent progeny.

Thus, in cattle and mice at least, it appears that telomere erosion generally does not occur in clones and is therefore unlikely to cause the long-term health and reduced life expectancy concerns raised by many recent reports.

The majority of (male) mice cloned from immature Sertoli cells died after approximately 500 days, which was around 50% of the lifespan in control mice. The causes of death were severe pneumonia and hepatic failure. It remains to be determined whether this is a general phenomenon with clones, but it appears to be both cell type and genotype specific, with other cloned mice having apparently normal life spans.

The mouse model has the advantage of a shorter generation interval and biological life span to screen for these effects. Whilst it is encouraging that some studies report normal health of four year old bovine clones, it is too early to detect if phenotypes with shorter life spans will also occur among livestock. Although an important issue, even if cloning were to shorten lifespan, it may be of little significance in agriculture.

In commercial beef production, for instance, cattle may be slaughtered at target live weight within two years, or in the dairy industry the average life span of a cow in the herd is only six years. In these examples, the productive life of farmed animals is substantially less than the biological limits for the species.

However, studies at AgResearch show that between weaning and four years of age, the annual mortality rate in cattle cloned from somatic cells is at least 8%. This is in marked contrast to the negligible mortality experienced with the offspring of clones and the typically accepted mortality of 2% to 3% per annum in conventional pastoral fanning.

Although the reasons for death amongst the clones are variable, and some potentially preventable, the main mortality factor beyond weaning is euthanasia due to musculoskeletal abnormalities.

This includes animals with severely contracted flexor tendons and those displaying chronic lameness, particularly in milking cows. This emphasises the point that any underlying frailties in cloned animals may not be fully revealed until the animals are stressed in some manner.

Again in mice, it was initially reported that females cloned from cumulus cells developed an increased body-weight phenotype, commencing eight to ten weeks after birth that was directly attributable to increased adipose tissue. However, this obese phenotype has also been recently observed in cloned mice produced from immature Sertoli cells and appears to occur at a greater frequency in agouti mouse strains. At present there is no indication for early onset obesity occurring in livestock.

Evidence of a compromised immune system is a clone phenotype noted in some species. Thymic aplasia has been documented in cloned cattle and lower levels of antibody production in cloned mice and of cytokines in cloned pigs are direct indicators of a reduced immune response.

This may increase their susceptibility to infection and disease. The incidence of enteritis, umbilical and respiratory infections are certainly increased in cloned livestock. However, others have reported normal characterisation of peripheral blood lymphocytes and normal responses to periodic infection in cloned cattle.

Assessment of animal behaviour and cognitive function provide an indicator of general physiological state and wellbeing. An examination of the behaviour of cumulus cell cloned mice revealed that there was a delay of 0.6 to 2.3 days in the first appearance of three out of ten pre-weaning developmental behaviours and milestones examined. However, subsequent tests on spatial learning, memory, activity level and motor skills were comparable to controls.

Trans-Generational Effects:

It is important to not only monitor the health of the clones but also their subsequent progeny derived following sexual reproduction. Offspring of male and female clones in a range of species have been produced following both natural mating and assisted sexual reproduction, such as artificial insemination, with a non-cloned partner. Conception, pregnancy, parturition and survival are all within normal ranges, as is the subsequent fertility of these offspring of clones.

More discriminatory, is the mating of cloned females with cloned males. With these matings in sheep, cattle and mice there is no evidence of the placental abnormalities and large birth weights recorded in the clone generation.

It has also been claimed that the obese phenotype observed in cumulus cell mouse clones is not heritable following mating with cloned males derived from fibroblasts of the same mouse strain.

However, it has not been reported whether the males of this strain also have the obese phenotype. If the obesity is truly non-heritable, then another generation of inbreeding would be required to exclude the possibility of a recessive genetic (or epigenetic) trait.

The most convincing evidence for the lack of transmission of any obvious deleterious recessive genetic or epigenetic trait has been provided following the mating of cloned male and cloned female mice (derived from XY and XO embryonic stem cells, respectively) obtained from the same cell line. The resulting offspring were phenotypically normal; lacking the foetal and placental overgrowth and open-eyelids-at-birth characteristic of their cloned parents.

The observations above, indicating that the clone associated phenotypes are not transmitted to offspring following sexual reproduction, implies that they are epigenetic in nature and that any errors in the surviving clones appear to be reset or corrected during gametogenesis. This is encouraging for the major application of cloning technology in agriculture; namely, the generation of cloned sires from progeny-tested, genetically elite males.

The cloning of elite sires means that their superior genes will be more widely disseminated following either increased semen production for artificial insemination or natural mating. Nonetheless, it is still possible that heritable genetic errors may be present in the clones.

Moreover, detailed molecular studies are required to confirm whether the necessary epigenetic modifications in gametes, zygotes and embryos derived from cloned parents are indeed restored to normal.

It is critical to investigate this phenomenon more thoroughly, as evidence exists for the germ line transmission of epigenetic states at various endogenous loci and in more artificial situations, following nuclear-cytoplasmic incompatibility. Additionally, at a practical level, it remains to be demonstrated that the daughters of cloned dairy sires, for instance, have a similar phenotypic performance to contemporary progeny of the original donor bull.

Livestock Production and Food Safety:

Few scientific reports regarding the production characteristics and food safety of somatic cell clones have been published to date. However, to address safety concerns, information has been provided to national regulatory agencies in a number of countries to demonstrate compositional equivalence of food products derived from cloned livestock.

As expected, given the current state of the technology, subtle epigenetic differences in somatic cell clones appear to increase the variation in animal performance between members of a clonal family. This variation in phenotype is anticipated to be greater than that between naturally occurring monozygotic twins.

Despite this, the composition of meat and milk from cloned cattle are within the normal ranges for these food products. This is further supported by recent data examining some of the constituents for which milk is an important dietary source.

The average levels of minerals, amino acids and vitamins from the milk of nine pastorally-fed cloned cows (with three cows each representing three different clonal families) were comparable to those from five control cow$, examined at a similar stage of lactation in springtime.