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Notes on the Construction of Transgene!
The designing of transgene varies grossly depending upon the type of gene of interest we are interested in. For example, metallothionein-l (MT-l) promoter is used for expression of rat growth hormone (rGH) in the liver under the signal of heavy metal ions.
Similarly, cloned bovine metallothionein-buffalo growth hormone transgene construct may be used for producing transgenic cattle for buffalo growth hormone. If the promoter from a metallothionein gene is attached to the coding sequence of, say, a growth hormone gene, the resultant transgene can be switched on simply by adding heavy metals to the drinking water of the transgenic animals.
This means that the production of growth hormone from the transgene can be turned on and off by a simple manipulation of the drinking water.
Alternatively, if the aim to have a transgene expressed in just one tissue, a promoter is chosen from a gene that is naturally expressed only in that tissue. For example, a transgene containing the promoter from a milk protein gene will be expressed only in mammary gland of the transgenic animal.
Pre-Implantation Analysis:
Reliable Screening of the embryo for transgene integration prior to transfer is essential because of very low transgenesis rate in farm animals. If the biopsy of in vitro produced embryo shows decrease in embryonic, then this can be compensated by transferring multiple embryos to each recipient.
Sperm Sexing:
The selection of a particular sex is used to produce offspring of the desired sex. It also offers a way of avoiding the expression of the disease in the offspring. It is also essential to sex embryo and only transfer the embryo of same sex to one recipient in order to avoid free martinism.
Also, since for mammary gland transgenesis females are to be produced, the sexing of the pre-implantation embryo is highly desirable. After pre-implantation analysis the female embryo which harbour transgene are transferred to the foster mother and allowed to complete the term.
Pre-implantation Embryo Nutrition:
The most important nutrients required by the developing mammalian pre-implantation embryo are carbohydrate, amino acid which provide energy. It also prevents cellular stress. Quantification of embryo energy metabolism is thus necessary.
Choice of the Host:
Before selecting host ore question need to be asked, whether the transgenic product is useful to the animal itself or it is to be expressed in host for commercial purposes. If the product is useful to the animal itself, then one can go straightway to the animal of choice. This has been demonstrated for the production of large number of animals harbouring growth hormone transgene.
If the transgenic animal is to be used as a bioreactor for the production of heterologous protein in some extracellular fluid, then the following criteria may be taken into consideration:
a. Animal should produce large volume of extracellular fluids,
b. Purification of heterologous protein from extracellular fluids should be relatively easy,
c. Have gestation and development periods of moderate length, and
d. There should not be adverse effects on the physiology of the transgenic animals.
Post-Birth Analysis:
Neonates are analysed for transgene and expression of the transgene. The new born transgenic animals are then screened for the presence of transgene (i.e., ear, tail and blood DNA) using southern blotting of the pure genomic DNA with subsequent probing using DNA probes from transgene construct. They are then mated with their transgene sibling to obtain homozygous transgenic animal.