This article throws light upon the three methods used for introducing a foreign gene into transgenic mice.
The three methods are: (1) Retroviral Vector Method (2) Microinjection Method and (3) Embryonic Stem Cell Method.
Methods for Introducing a Foreign Gene:
The transfer of small pieces (8 kb) of DNA can be effectively carried out by retroviruses. This method, however, is unsuitable for transfer of larger genes. Further, even for small genes, there is a risk of losing some regulatory sequences.
Above all, the biggest drawback is the risk of retroviral contamination in the products (particularly in foods for human consumption), obtained from transgenic animals. Because of these limitations, the retroviral vector method is not in regular use for trans-genesis.
Method # 2. Microinjection Method:
The introduction of DNA by microinjection method involves the following steps (Fig. 41.1).
1. The young virgin female mice (4-5 weeks age) are subjected to superovulation. This is achieved by administration of follicle-stimulating hormone (pregnant mare’s serum), followed by (2 days later) human chorionic gonadotropin. The super ovulated mouse produces 30-35 eggs (instead of normal 5-10 eggs).
2. The above female mice are mated with males and sacrificed on the following day. The fertilized eggs are removed from the fallopian tubes.
3. By micromanipulation using a microinjection needle and a holding pipette, the DNA is injected into the male pro-nucleus of the fertilized egg. Adequate care must be taken to ensure that while the elastic nuclear membrane is punctured, the needle does not touch the nucleoli. A dissection microscope can be used for identifying the male pro-nucleus (larger in size) and for microinjection.
4. The eggs with the transgenes are kept overnight in an incubator to develop to a 2-cell stage. These eggs are then implanted micro surgically into a foster mother i.e., pseudo- mouse pregnant female mouse which has been mated the previous might with vasectomized (or infertile) male. The foster mother can deliver pups after 3 weeks of implantation.
The presence of transgene in the pups can be identified by polymerase chain reaction or Southern blot hybridization. The mouse carrying the foreign gene is the transgenic founder from which pure transgenic lines can be established.
The microinjection method involves several steps and none of them is 100% efficient for any animal to develop into transgenic animal. In case of mouse, it was found that about 65% of the fertilized eggs survive microinjection procedure, about 25% of the implanted eggs develop into pups, and only 25% of them are transgenic. Thus, if one starts with 1000 fertilized eggs, only 30 to 50 transgenic pups may be produced i.e., 3-5% of the inoculated eggs develop into transgenic animals.
Limitations of microinjection method:
Besides the low efficiency there are some other disadvantages in this technique. The foreign DNA randomly integrates into the host genome. Sometimes, even many pieces of DNA get incorporated at a single site. Further, transgenes may not be expressed at all or sometimes under expressed, or even overexpressed.
All these processes will disturb the normal physiology of the transgenic animal. In addition, microinjection procedures are time consuming, costly and labour intensive. Despite all these limitations, this technique is routinely used for producing transgenic animals.
Method # 3. Embryonic Stem Cell Method:
Cells from the inner cell mass of the blastocyst stage of a developing mouse embryo can proliferate in cell culture. These cells, referred to as pluripotent embryonic stem (ES) cells, are capable of differentiating into other types of cells (including germ line cells) when transferred to another blastocyst embryo.
The embryonic stem cell technology basically involves the introduction of a foreign DNA into ES cell (Fig. 41.2). Embryonic stem cells in culture can be subjected to genetic manipulations without changing their plutipotency. Foreign DNA can be introduced into ES cells by electroporation or microinjection. The desired genetically engineered cells with transgene can be identified by a selection procedure using a marker gene or PCR analysis (described below).
The transfected cells can be cultured, introduced (by microinjection) into blastocyst and then implanted into foster mother (i.e., pseudo pregnant female mouse). By this way, transgenic founder mice are produced. Transgenic lines can be established by suitable breeding strategies of the founder mice.
Selection of transgene containing cells:
Several strategies have been developed for the selection of transgene containing cells. The important ones are briefly described.
Selection by use of marker gene coding for thymidine kinase:
It is worthwhile to know the role of thymidine kinase to understand its utility as a marker gene. There are two pathways for the synthesis of deoxyribonucleotides (dATP, dGTP, dCTP and dTTP), the basic units of DNA structure.
One is the salvage pathway that recycles the degraded nitrogenous bases formed from DNA. The other alternate pathway is an endogenous synthetic pathway from different precursors (glycine, aspartate, glutamine, methyl tetrahydrofolate etc.). The enzyme thymidine kinase (TK) is involved in the salvage pathway. TK phosphorylates thymidine to produce thymidine monophosphate (dTMP) which is finally converted to thymidine triphosphate (dTTP).
The gene that encodes the enzyme thymidine kinase can be used as a marker to determine whether the transgene has been inserted. This is illustrated in Fig. 41.3. The mammalian cells are capable of synthesizing dTTP by salvage pathway and endogenous synthetic pathway. The cells lacking TK gene cannot produce dTTP.
If such cells are cultured in a HAT medium containing Ziypoxanthine (H), aminopterine (A) and thymidine (T), they cannot grow and therefore die. This is because thymidine cannot be utilized in the salvage pathway due to lack the enzyme thymidine kinase. Further, aminopterine blocks the endogenous pathway (by inhibiting the enzyme dihydrofolate reductase, required for one carbon metabolism).
If a transgene is joined to a TK gene, inserted into a mammalian cell (TK–) and then the cells can grow in HAT medium. This is possible only if the TK gene is incorporated into the mammalian cells. And logically, the cells that survive in HAT medium carry the transgene. In this fashion, thymidine kinase can be effectively used as a marker gene.
There are other enzymes that serve as markers for identifying transgene insertion. These include dihydrofolate reductase (resistant to methotrexate) and neomycin phosphotransferase (resistant to antibiotic G418) and PCR analysis for selecting transgene containing cells. The last one is a more direct and recent method, and is successfully used for detecting transgene containing cells.
Promoter sequence to facilitate trans-genesis:
In the early experiments on trans-genesis, the mouse metallothionein (MMT) gene promoter was used. MMT gene encodes for a metal binding protein that is involved in metal homeostasis. The foreign gene (i.e., a rat growth hormone gene) can be linked to a promoter sequence of MMT.
By doing so, the promoter switches on the growth hormone gene when the MMT is activated by a metal in the environment (e.g., cadmium). Thus, the metal inducer (Cd) can stimulate the promoter (MMT promoter) to facilitate the transgene (growth hormone gene) to express. Therefore, addition of cadmium triggers the growth hormone production.
By inserting a transgene (foreign gene) into a chromosome, a new function is introduced while producing transgenic animals (described above). On the other hand, in a process referred to as gene knockout an existing function can be blocked by destroying a specific gene. The target gene disruption can be carried out by incorporating a DNA sequence, usually a selectable marker gene (smg) into the coding region.
This is depicted in Fig. 41.4. The chromosome carrying the target gene (with 4 exons) with flanking sequences is subjected to homologous recombination with a vector carrying a selectable marker gene. The homologous recombination results in gene knockout i.e., disruption of the target gene.
In the gene knockout, the loss-of-function occurs in transgenic animals. This is in contrast to gain of function that takes place by introducing a foreign gene.
Applications of gene knockout:
Disruption of the target genes (gene knockout) is important for understanding the development and physiological consequences in an organism. Further, the biochemical and pathological basis of several human diseases can be appropriately understood by inactivating specific genes.
About 200 knockout mice have been so far created to serve as animal models for the study of a large number of human abnormalities and disorders. In fact, knockout mouse which lack genes for a single organ or organ system can be produced
Yeast Artificial Chromosome in Trans-genesis:
In the conventional trans-genesis, small sized transgenes (≤ 20 kb) are transferred through vectors. This involves a risk of losing some important sequences including the regulatory ones. Further, several genes exist as complexes and are too large to be handled by conventional vectors. Yeast artificial chromosomes (YACs) can carry large-sized genes (size range 100-1,000 kb), and are effectively used in trans-genesis. By using microinjection technique or transfection of ES cells with YACs, transgenic mice have been produced.
Selected examples are cited below:
1. YACs are used to carry human β-globin gene complex (5 genes with 250 kb size) and produce transgenic mice.
2. YACs are employed for producing human antibodies in transgenic mice. Synthesis of antibodies is very complex process. YACs carrying varying sizes of DNA sequences (800-1,000 kb YACs) have been employed to generate different antibodies.