VPB 321: Animal Biotechnology (2+1)

MICROMANIPULATION

• In 1966, Lin described the technique of micormanipulating and injecting a mouse egg
• Subsequently, transgenic animals have been produced by introduction of foreign genes at the pronuclear stages of fertilized, one-cell zygotes
• Most of the successes have been with mouse and recently successful production of transgenic rabbit, pig, sheep and goat have been shown
• This technique is a powerful tool for studying gene regulation and physiological functions of gene products in a normal host environment
• Micromanipulation is the technique whereby sperm, eggs and embryos can be handled on an inverted microscope stage, performing minute procedures at the microscopic level via joysticks that hydraulically operate glass microtools.
• The injection of a single sperm into the cytoplasm of the oocyte, or intracytoplasmic sperm injection (ICSI), provided a satisfactory solution to the problems of the assisted fertilization techniques developed earlier.
• In this procedure, a single sperm is first immobilized by touching the sperm tail with an injection pipette (inner diameter 5–7 μm). The injection pipette picks up the immobilized sperm, pierces the ZP and oolemma, and delivers the sperm inside the oocyte cytoplasm.
• In 1976 using hamsters as a model, Uehara and Yanagimachi were probably the first to report the injection of sperm into oocyte cytoplasm (ooplasm).
• It was later attempted on rabbit and human oocytes, although the first successful human pregnancy was not reported until 1992 by the Free University of Brussels’ group in Belgium.
• Micromanipulation technology has enabled the reproductive biologist to overcome inefficient steps in mammalian fertilization, the production of chimeric animals through blastocyst injection with embryonic stem (ES) cells and the introduction of specific genes into the genomes of domestic and laboratory animals.
• This technology has also been used for the production of cloned animals and ES cell lineages from cloned embryos, using nuclear transfer. Moreover, micromanipulation is also used for microsurgical embryo biopsy to study the basic developmental biology of embryos during preimplantation development.