Cloned cat deficiencies: Protein changes account for premature deaths

Poor cloning survival rates

One of the established animal cloning techniques is somatic cell nuclear transfer (SCNT) which has been applied to the successful reproduction of sheep, mice, goats, cattle, dogs and cats. However, despite the apparent success, the survival rate of cloned animals is disappointingly low. The difficulties have been attributed to abnormal gene expression in cloned placentas which results in the loss of foetuses or the rapid death of newly born offspring.

During SCNT, the nucleus is removed from the somatic cell of an adult animal and used to replace the nucleus of an egg cell. The somatic cell nucleus is reprogrammed by the host cell and cell division begins, resulting in the formation of an embryo. It has a complete set of chromosomes from the somatic cell, so grows into an identical genetic replica of the adult from which the cell nucleus was taken.

Well, that is the theory but clearly, somewhere along the line, the genetic make-up is disturbed in many of these cloned embryos, leading to developmental problems. The resulting placental problems can include increased placenta weight, DNA methylation and protein modification.

A team of Korean scientists has been cloning cats since 2005 but their efforts have also been blighted by the premature death of many kittens. Il-Keun Kong and colleagues from Gyeongsang National University, Jinju National University and Dong-eui University describe this as the main factor that limits efficiency.

Cats are genetically quite close to humans and more than 200 feline diseases closely resemble human diseases, so there is good reason to undertake genetic studies on them. Cloning cats with the same diseases as humans would allow scientists to conduct experiments that are not possible with human patients.

An additional reason for trying to perfect cloning cats is to help restore endangered species like Siberian tigers, lynxes and jaguars.

So, Kong and his team have undertaken a proteomics study of the placentas of cloned cats to try and establish the unregulated changes that are occurring as a result of cloning.

Placental proteins unbalanced

In the first instance, placentas were harvested from normal cats giving birth by caesarean section or vaginal delivery following natural mating and the proteins were extracted for comparison by 2D gel electrophoresis. The gel patterns for the two groups were identical, showing that the method of delivery does not affect the protein composition during natural conception.

The protein patterns from cloned placentas during birth by caesarean section did show some variations, with 13 up-regulated compared with the natural controls and 7 down-regulated. The proteins involved were identified by mass spectrometry and their functions discussed in terms of the hampered development of placentas during cloned pregnancy.

One of the key proteins that was down-regulated was prohibitin, which regulates apoptosis, also know as programmed cell death. Its change in abundance was linked with an increase in reactive oxygen species (ROS) which damage placental tissue and are associated with senescence (cell aging) and apoptosis.

The fall in prohibitin was accompanied by an increase in the level of cathepsin D, a protein which is also related to apoptosis and is found in nearly all animal cells. The rise in cathepsin D was matched by increases in cytochrome c, caspase-3, caspase-9 and Bax, also known as Bcl-2-associated X protein. These proteins are all involved in the path that leads from cathepsin D to apoptosis. In addition, p53, a biomarker of apoptoisis, was up-regulated in the cloned placentas.

So, the abnormal production of prohibitin and cathepsin D could be linked to the increased death rates of newborn kittens by the rise in reactive oxygen species, leading to increased mortality.

Various other proteins were also differentially regulated in the cloned placentas and might be linked to the reduced viability of cloned cat foetuses. For instance, the glycolytic enzyme triosephosphate isomerase was down-regulated, a state that has been associated with neurological dysfunction and early childhood death.

Increased levels of heat shock protein 27 were linked to a response to oxidative stress, possibly induced by the ROS. The up-regulation of actin regulatory protein CAP-G might be linked to easier migration and metastasis of tumour cells during hypoxia. Increased tropomysin-4 is a biomarker for growth and repair following injury, stress or disease.

Taken together, the results give a strong indication that the low cloning efficiency in cats is a direct result of abnormal protein production in the placentas. A closer examination of the key factors involved could lead to strategies for adjusting the protein expression during foetal development and placental growth that would improve survival rates.

If successful, it would help to protect endangered species by providing cloned offspring that had a significantly increased chance of survival.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.