Techniques of Genetic Engineering

Microinjection is a technique of delivering foreign DNA into a living cell (a cell, egg, oocyte, embryos of animals) through a glass micropipette. One end of a glass micropipette is heated until the glass becomes somewhat liquified. It is quickly stretched which forms a very fine tip at the heated end. The tip of the pipette attains to about 0.5 mm diameter which resembles an injection needle. The process of delivering foreign DNA is done under a powerful microscope. Cells to be microinjected are placed in a container (Fig. 4.15). 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.

  A method of microinjection of DNA preparation in egg.  

Fig. 4.15. A method of microinjection of DNA preparation in egg.


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. Some of the endogenous pattern of gene regulation during development has been characterized (Wickens and Laskey, 1981). 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. There are many examples where different types of animal cells have been microinjected and successfully transferred.
Rubin and Spradling (1982) for the first time introduced Drosophila gene for xanthine dehydrogenase into a P-element (parental clement) which was microinjected with an intact helper P-element into embryo deficient for this gene. Such embryos later on developed flies with rosy colored eyes than mosaic eyes as in the first parental generation.
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 the 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 of 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 (Fig. 4.16).
» Gene cloning in prokaryotes

» Isolation of DNA to be cloned

» Insertion of DNA fragment into vector 

» Use of restriction Linkers

» Use of homopolymer tails

» Transfer of recombinant DNA into bacterial cells

» Selection of clones

» Colony hybridization techniques

» In vitro translation technique

» Immunological tests

» Blotting Techniques

» Recovery of cells

» Expression of cloned DNA

» Shine-Dalgano sequence

» Expression vectors
» Gene cloning in eukaryotes

» Plant cells

» Yeasts

» Filamentous fungi

» Agrobacterium plasmids

» Plant cell transformation

» Plant cell transformation by ultrasonication

» Liposome mediated gene transfer

» Animal cell  

» Animal viruses

» Electroporation

» Particle bombardment

» Microinjection

» Direct transformation
» Site directed mutagenesis

» Methods of mutagenesis

In another experiment ( Palmiter et al 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
The term in vitro means in glass or in artificial condition, and IVF refers to the fact that fertilization of egg by sperm occurs in glassware.
  Production of transgenic mice; MT, methionine.

Fig. 4.16. Production of transgenic mice; MT, methionine.


IVF technology in farm animals
Nowadays, exogenous hormones have been developed through naturally and recombinant DNA technology which are used to induce superovulation in farm animals. Superovulation is a phenomenon of producing greater than the normal number of eggs through hormonal treatment by a single female at a time.

A normal cow produces one or two eggs during an ovulatory period, whereas the same can produce 8-10 eggs when subjected to superovulation. Therefore, through normal reproduction, an animal produces about 4-5 offsprings in her life, while through IVF technology the same can produce 50-80 offsprings in her life. For a detailed description see Manipulation of Reproduction and Transgenic Animals.

IVF technology in humans
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 in 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 India, there are nine centers like Mumbai, Kolata, Chenai, Delhi and others assisting the childless couples.
In addition to ZIFT and GIFT, the microinjection technique is also applied in oligospermic patients. Here, one sperm is directly injected into an egg to facilitate fertilization.
Problems related to test tube babies
Although the IVF technology is a boon to childless couples, yet there are several problems related to it, if it becomes commercialized. These problems may be religious, ethical, emotional or political. For example, the Catholic Church does not approve IVF technique as it proclaims that conception should never be taken out of the body. The GIFT is acceptable, while ZIFT is not. Muslim countries like Malaysia believe the sperm donation as immoral. Children borne of donated sperms are considered illegitimate. However, there is no controversy in India so far. A sperm bank has been set up in New Delhi in January, 1994 to help the childless couples.

One of the emotional problems is the unused extra embryos. Whether they will be thrown away or implanted in surrogate mothers. The first one is the moral question. The surrogate mothers act as animal incubator and deliver baby after the normal gestation. They don't contribute the genetic materials as it come from the donors. Therefore, the surrogate mothers could be commercialized. The child will not be their real one. Will the children borne through donated egg/sperm be given social or religious recognition? Will the children be known of their biological parents? Will their parents accept them?