DNA Microinjection | IVF technology | ICSI Procedure
Microinjection is a technique of delivering foreign DNA into a living cell through a glass micropipette. One end of a glass micropipette is heated until the glass becomes somewhat liquefied. It is quickly stretched which forms a very fine tip at the heated end. The tip of the pipette becomes about 0.5 mm in diameter which resembles an injection needle. Delivery of foreign DNA is done under a powerful microscope. Cells to be microinjected are placed in a container. A holding pipette is placed in the field of view of the microscope. The holding pipette holds a target cell at the tip when gently sucked. The tip of the micropipette is injected through the membrane of the cell. Contents of the needle are delivered into the cytoplasm and the empty needle is taken out.
Xenopus oocytes have been widely used for the study of transcription by microinjection because oocytes contain between 6,000 and 100,000 or more RNA polymerase molecules than somatic cells. Microinjection is technically easy because of large size of oocytes. The injected DNA integrates randomly with nuclear DNA and its expression could be possible only when the foreign DNA is attached to a suitable promoter sequence.
Production of Transgenic Animals
In 1982, R. D. Palmiter of Washington University and R. L. Brinter of Pennsylvanian University isolated the rabbit growth hormone (p-globin) gene, human growth hormone (i-globin) gene as well as thymidine kinase gene and linked separately to the promoter region of mouse associated with the metallothionein I gene (a gene which encodes a metal binding protein). This was joined to pBR322 plasmid to produce the recombinant plasmids. Mature eggs from adult mouse were recovered surgically and fertilized with sperms in vitro. Immediately after fertilized eggs were microinjected with recombinant plasmids before the sperm and egg nuclei have fused to form a diploid zygote. The plasmids generally combine homologously with each other within the egg forming a long repeated concatemer which then integrates randomly to give repeated genes at a single chromosomal site. The engineered embryos were then implanted into the uterus of a host mouse mother for further development. The resulting mice are called ‘transgenic mice since part genome comes from another genetically unrelated organism. Due to introduction of foreign gene before nuclear fusion, chromosomal integration takes place early and progeny contains new genes. Size and body weight of progenies were extremely larger than the normal ones.
In another experiment (Palmiter, 1982) injected mouse embryos with a DNA fragment containing the rat growth hormone gene fused to the promoter region of the mouse metallothionein I gene. In this experiment, linear DNA fragments were used rather than plasmids because these integrate more efficiently into the mouse chromosomes. Consequently, 21 mice were produced, among them seven contained the fusion gene. Six were two fold larger in body weight than the others. The level of growth hormones increased many times (between 200-800 times) more than the control. In addition, the level of growth hormone mRNA was also increased in liver cells. Similarly, many transgenic animals such as sheep, goat, pigs, rabbit, etc. have also been produced through microinjection technique.
In-vitro fertilization (IVF) technology
IVF technology was pioneered in humans by Prof. Robert Winston. The same technique was used by P. Steptoe and R.Edwards to produce world’s first test tube female baby, Louise J. Brown, on July 25, 1978. Since then more than 25,000 babies have been produced so far.
Earlier eggs were recovered from the patient’s ovary by using laparoscope. A small incision is made just below the navel and the laparoscope is introduced. Eggs are removed with a hollow needle. Generally, only a single egg can be procured at a time after certain period in natural way. But through superovulation more eggs can be obtained at a time.
The hormone is injected daily for about 28 days. Sometimes side effects may take place. The eggs are kept in a special fluid and examined microscopically for any defects. Eggs are transferred into a Petri dish containing fresh semen. The gametes take 12-15 hours to fertilize. After fertilization zygote is kept another fluid at the body temperature. Cell division is observed regularly. When embryo reaches to blastocyst stage, the last stage of growth, it is implanted into uterus. It is not necessary that all the implanted embryos will grow. There are many complications related to it after implantation. Therefore, to get success generally three embryos are transferred into the uterus at a time. This results in birth of one, two or three babies based on success. This process is called as zygote intrafallopian transfer (ZIFT). There is another technique where eggs and sperms are placed in fallopian tube to facilitate fertilization. This is known as gamete intrafallopian transfer (GIFT).
In addition to ZIFT and GIFT, the microinjection technique is also applied in oligospermic patients. Here, one sperm is directly injected into an egg (ICSI) to facilitate fertilization.
In-Vitro Fertilisation with intracytoplasmic sperm injection (ICSI)
In Vitro Fertilisation with ICSI is an assisted reproduction procedure in which a single sperm cell is injected by micro-injection directly into an egg. Once fertilised, the egg becomes a pre-embryo and is transferred into the uterus to continue its development.
This technique involves the insemination of an egg by a sperm microinjection. The steps before and after insemination are exactly as in a classical IVF without ICSI, the only change is the insemination technique. To perform ICSI only one sperm is needed per egg, while in a classic IVF without ICSI between 50,000 and 100,000 are needed. Once fertilised, the egg becomes a pre-embryo and is transferred to the uterus to continue development.
ICSI was developed in 1992 to treat cases of male infertility or abnormalities in the sperm: azoospermia (no sperm), oligozoospermia (low concentration of sperm), asthenozoospermia (low motility) or teratozoospermia (poor sperm morphology) and since then there have been major advances made in the treatment of infertility of male origin. Today it is routinely used.
Monitoring and stimulating the ovaries
The ovaries are stimulated by administering hormones (FSH, Follicle Stimulating Hormone and in some cases, Luteinizing Hormone, LH) and the cycle is monitored using scans until the follicles reach the correct number and size (only one follicle is needed for this technique). At this point, another hormone is administered that imitates LH, the hormone that naturally triggers ovulation (LH or Luteinizing Hormone), and this results in an egg being released.
Egg harvesting and in vitro fertilisation
Harvesting the eggs is done by inserting a needle into an ovarian follicle and aspirating it. The procedure is done while the woman is under sedation. Once they have been removed, the eggs are stored in a culture dish while the semen is prepared to separate out the motile sperm. Then the eggs are denuded, which means that the cells on the surface are removed, and a single sperm is injected into each one.
The day after harvesting and the ICSI procedure we will know how many eggs have been fertilised. Over the next 2 or 3 days, these fertilised eggs become pre-embryos ready to be transferred to the uterus. On transfer day, the pre-embryos showing the best signs of developing are selected. The law permits us to transfer up to 3 pre-embryos, but the average number is 2.
The pre-embryos are placed in a thin catheter and the gynaecologist inserts the eggs deep inside the uterus. No anesthetic is required for this procedure. Of the transferred pre-embryos, normally only one will implant, but bear in mind that sometimes more than one may implant, resulting in multiple pregnancy.
The pre-embryos that have not been transferred are frozen in liquid nitrogen (this type of cryopreservation is known as Vitrification) and they are then carefully labelled and stored in the embryo bank. These pre-embryos can be used in subsequent cycles if pregnancy is not achieved on the first attempt. Evidently, the treatment for preparing the uterus for the transfer of frozen embryos is much simpler as there is no need to stimulate the follicles and harvest the eggs.
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