Animal Cloning by Somatic Cell Nuclear Transfer
Animal cloning refers to the production of genetically identical copies of individual animals by means of nuclear transfer. Animal cloning involves advanced methods of microsurgery, embryo culture, and transfer into recipients (surrogate mothers).
Animal cloning is a technique by which a nucleus from a cell of the donor animal is inserted into an enucleated oocyte, and reconstructed oocyte is allowed to grow upto embryonic development and embryos are transferred into a recipient animal. The method is therefore also known as somatic cell nuclear transfer (SCNT).
Fig. 24.6 Sexing of in vitro fertilized bovine embryos by FISH. Y chromosomal probe of Bos taurus (BtY2-L1) was detected by FITC- conjugated anti-avidin antibody (yellow). Propidium iodide (PI) was used for counterstaining the spread. (a) Metaphase nuclei of male-
putative blastomeres (arrow showing binding of Y-specific probe). (b) Metaphase nuclei of female-putative blastomeres (Y-specific probe is not bound). (Source: Lee et al. 2004)
Fig. 24.7 Timeline of the development of live offspring of farm animals. (a) Traditional cloning method and (b) hand-guided cloning method
Based on the method of enucleation process animal cloning may be either traditional cloning (TC) or hand-made cloning (HMC). TC uses micromanipulator apparatus for enucleation event, whereas HMC is a more advanced procedure. In HMC, enucleation of zona-free mammalian oocytes is performed with sharp metal blades for bisection of zona- free oocytes under stereomicroscope or density gradient centrifugation or chemicals.
24.8.1 Milestones
Briggs and King transferred nuclei from early cleavage stage embryos to enucleated eggs of the North American leopard frog Rana pipiens and tadpoles developed.
Comparatively large size of the eggs makes micromanipulation easier in amphibians. Figure 24.7a, b depicts the timeline of the development of SCNT animals both by TC and HMC methods.24.8.1.1 Hand-Made Cloning (HMC) Method
Figure 24.8 represents schematically the different steps of HMC method.
24.8.1.1.1 Preparation of Donor Somatic Cell or Nuclei
Sources of donor cells in case of HMC vary from species to species and conditions of the nuclear reprograming procedure. Depending on the type of donor cells used in HMC technique blastocyst production rate varies. Adult and fetal fibroblast cells are commonly used as donor cells in farm animals like cow, buffaloes, sheep, and goat. Apart from those, cells of different origins like pronuclear stage embryos, embryonic blastomeres, cumulus cells, granulosa cells, embryonic stem cells, lymphocytes, milk somatic cells, and urine epithelial cells have been used efficiently during HMC process in different species. The donor cells must be quiescent at G0 or arrested at G1 phase for optimum efficiency of cloning process. HMC with donor cells of S and G2/M phases cause premature chromosome condensation leading to chromosome pulverization and chromosomal aneuploidy, respectively which ultimately lead to poor cloning efficiency. Synchronization of cell cycle stage of donor cells is performed by treatment with chemicals like cycloheximide, DMSO, and roscovitine or serum starvation or contact inhibition by cell confluence.
Fig. 24.8 Steps of hand-guided cloning. Recipient and donor cells are prepared. Protrusion cone containing the oocyte nucleus is enucleated. Donor cells and enucleated oocytes are fused by electrical pulse. Finally, the reconstructed embryos are cultured for 6-7 days before transfer to the recipient animals
24.8.1.1.2 Recipient Oocyte Selection, In Vitro Maturation, and Enucleation
Various sources of oocytes are used for HMC method.
Oocytes may be obtained from live animals by ultrasound- guided ovum pick-up (OPU) or laparoscopic technique following hormonal stimulation of animals. Also, abattoir ovaries from nonstimulated females are also used for HMC. Good quality oocytes are selected on the basis of compact cumulus-oocyte complex (COC), and homogeneous ooplasm. In vitro maturation of oocytes is carried out for 22-24 h in a maturation medium containing the hormones like FSH, LH, and estradiol. After that, hyaluronidase is used to remove cumulus cells, pronase is used to remove zona, and zona-free oocytes are processed for enucleation. Enucleation of oocytes is done by cutting the polar body (protrusion cone) with the help of a microblade or chemically by the use of demecolcine, nocodazole, etoposide, caffeine, MG132, etc., or density gradient centrifugation. After bisection, a portion of oocyte with chromatin is known as karyoplast and that without chromatin is known as cytoplasts or hemi-cytoplasts (also known as demioocyte). Cytoplasts are screened by fluorescent staining and selected for embryo reconstruction purpose.24.8.1.1.2 Reconstruction of Embryos
In this step, demioocytes are exposed to phytohemagglutinin (PHA) for a few seconds. PHA makes a sticky layer on the surface of the demioocytes. This helps in easy attachment of the donor cell on its surface to form a couplet. As there occurs substantial loss of cytoplasm during the dissection of oocyte, another demioocyte is attached to the couplet to compensate for the loss of cytoplasm. A demioocyte-donor cell couplet and a single demioocyte are aligned in a BTX fusion chamber connected to an electrofusion device and a single low-voltage AC pulse in an electrofusion medium is delivered. Alternatively, two separate electrofusion events may be performed in the reconstruction process, initially during fusion of demioocyte-donor cell couplet fusion and second during couplet-demioocyte fusion. After electrofusion, chemical activation of rebuilt clone embryos is done with calcium ionophore and N-6 dimethylaminopurine (6-DMAP).
24.8.1.1.3 In Vitro Culture of Embryos and Transfer
HMC embryos are cultured in vitro individually in microwells using the well-of-the-well (WOW) system.
Activated HMC embryos are placed in each microwell which ensures three-dimensional blastomere arrangements in zona-free embryos. Embryonic quality and stages are assessed at definite interval of upto 7-8 days. Finally, good quality embryos are selected and transferred aseptically in the uterus of animals after 7-8 days.24.8.1.2 Traditional Cloning method
Traditional cloning methods employ micromanipulator apparatus which consists of a contrast optical system, stereomicroscope, micromanipulator, and microinjectors. Following steps are followed for the classical method of SCNT:
24.8.1.2.1 Enucleation
Enucleation of oocytes is done when the cell cycle phase of the oocyte is stalled at Metaphase II stage. During this stage, chromosomes remain condensed in the form of metaphase plate or meiotic spindle near the protruded polar body. Enucleation is performed in a small cohort of oocytes, usually with 10-20 oocytes at a time. The oocyte, polar body, and enucleation pipette are perfectly aligned for the removal of the metaphase chromosome. For optimum alignment, the oocyte is held gently by the holding pipette and the enucleation pipette is used to rotate the egg appropriately to bring the inner zona surface, polar body, and pipette tip in the same focal plane.
After securing the proper alignment, the holding pipette is made tighter and the enucleation pipette is introduced into the zona pellucida, piercing the oocyte membrane, adjacent to the metaphase plate.
A small amount of cytoplasm and polar body is gently sucked into the enucleation pipette. Enucleated oocyte is transferred from the micromanipulation chamber to the culture medium. Epi-illumination with Hoechst 33342 dye is done to confirm the successful enucleation.
Hoechst 33342 dye is done to confirm the successful enucleation and finally transferred to the culture medium. For preventing damage of the oocyte membrane during the process of piercing by enucleation pipette Cytochalasin B is used in the micromanipulation drop that destabilizes the actin cytoskeleton.
24.8.1.2.2 Donor Cell Preparation
Donor cells are harvested and prepared as described in HMC. Following harvesting cells are transferred into a fresh manipulation drop containing enucleated oocytes. Several cells are loaded into the cell transfer pipette at a time to hasten the process. Cells are lodged in the perivitelline space by placing the oocyte in such a manner that the slit in the zona pellucida formed during enucleation is close to the tip of the cell transfer pipette. Within the zona, the cell is transferred by pushing it out of the pipette with the microinjector, ensuring proper contact between the cell and the oocyte membrane.
24.8.1.2.3 Fusion of Donor Cell-Oocyte Couplet
Electrical stimulation is the most popular method of fusion of donor cell-oocyte couplet. Other methods like the use of polyethylene glycol (PEG), or Sendai virus have also been used by several researchers in different species. Electrofusion medium is a non-ionic, slightly hypotonic medium. A common electrofusion medium consists of 0.3 M mannitol, 0.1 mM MgSO4, and 0.05 mM CaCl2 which vary from species to species. Mannitol determines the osmolarity of the medium. Divalent magnesium cation helps to maintain membrane contact between the cell and oocyte calcium ion provides activation stimulus at the time of fusion. Fusion chambers of BTX instruments and temperature of the fusion medium are key factors that influence the efficiency of cloning experiment. A four-well plate is used for fusion purpose. Well 1 contains manipulation medium, Well 2 contains a 1:1 mixture of manipulation medium and fusion medium, Well 3 contains fusion medium, and Well 4 contains manipulation medium. From the manipulation, microscope couplets are moved to Well 1, then to Wells 2 and 3, where they settle to the bottom of each well. Once fused, they are placed into Well 4. Finally, they are placed in a culture medium and kept in the incubator. Chemical activation of reconstructed clone embryos is done with calcium ionophore and N-6 dimethylaminopurine (6-DMAP).
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Improving Animal Cloning Efficiency by Epigenetic Reprogramming
Since the birth of Dolly 25 years ago significant improvement has occurred in the efficiency of cloning procedure. Still the proportion of cloned embryos that develop to full term remains very low, greatly limiting the application of SCNT technology. The major reason behind that is incomplete epigenetic reprogramming during cloned embryo development. Epigenetic modifications are heritable changes in gene expression without alterations in genomic DNA sequences. Several epigenetic changes like DNA methylation, histone modification, genomic imprinting, and X chromosome inactivation (XCI) occur during the progression from fertilized oocyte to differentiated embryo and they also play a key role in embryo development following SCNT. Present research on SCNT focuses to improve epigenetic reconstruction in cloned embryos and several strategies have been devised so far. Improving DNA methylation reprogramming by DNA demethylation reagents and Dnmts knockdown has successfully ameliorated genome DNA methylation and histone modification in cloned embryos. Another method for improving the development competence of cloned embryos is to modify histone marks. Trichostatin A (a class I and II Histone deacetylase or Hdac inhibitor), scriptaid (a low-toxicity synthetic Hdac inhibitor), and valproic acid all increase histone acetylation, particularly H3K9ac and H3K14ac, boost gene expression levels in cloned embryos, and hence promote SCNT-mediated nuclear reprograming.
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