Some procedures applied for gene cloning in eukaryotes are described in this context: 1. Transformation of Filamentous Fungi 2. Gene Transfer through Ti-plasmid of Agrobacterium 3. Gene Transfer through Gene Gun 4. Gene Bank or Genomic Library.
1. Transformation of Filamentous Fungi:
Like bacteria, transformation of filamentous fungi has also been demonstrated. Hinnen (1978) first described transformation of protoplasts of Saccharomyces cerevisiae by E. coli plasmid. Later on transformation was demonstrated in Neurospora crassa.
The situation of filamentous fungi is different from the others as there is no convincing evidence for autonomously replicating plasmids, but sequences having functions similar to ARS of shuttle vector have been isolated.
There are several examples where transformation of fungal protoplasts has been carried out such as Aspergillus nidulans, A. niger, A. oryzae, Cephalosporium acremonium, Coprinus cinereus, Glomerella cingulata, Mucor sp., Penicillium chrysogenum, Sepotoria nodorum, etc.
Adopting this approach many mammalian genes have been expressed in S. cerevisiae and aspergilli through expression vector (i.e. vector which include sequences promoting high level of transcription of the gene and secretion of the gene products). Gwynne (1987) have found the secretion of about 1g/litre of human interferon from transformed A. nidulans mycelia.
2. Gene Transfer through Ti-Plasmid of Agrobacterium:
The Ti-plasmids of Agrobacterium tumifaciens (see types of plasmids). The T-DNA of Ti-plasmid is excised and nos and ops genes are replaced with a foreign DNA of known function.
The opine synthetase is retained to act as promoter for expression of inserted DNA. Murai (1983) have succeeded in introducing a gene for bean plant storage protein (phaseolin) into the cells of sunflower plant. They integrated phaseolin gene with Ti-plasmid and transformed A. tumifaciens cells lacking Ti-plasmid. The transformed bacterial cells infected the plant and delivered phaseolin gene that expressed normally.
3. Gene Transfer through Gene Gun:
In 1987, Prof. Stanford and co-workers at Cornell University (U.S.A.) first developed the gene gun that operates like shotgun. It shoots foreign DNA into plant cells or tissues at a very high speed. This technique is also known as particle bombardment, particle gun method, biolistic process, micro-projectile bombardment or particle acceleration.
This technique is applicable for those plants which hardly regenerate and do not show sufficient response to gene transfer through Agrobacterium for example, rice, wheat, com, sorghum, chickpea and pigeon-pea. Success has been achieved in shooting the missing DNA into the chloroplast of Chlamydomonas and mitochondria of yeast.
This apparatus consists of chamber connected to an outlet to create vacuum (Fig. 11.10). A 0.22 caliber blank shell shoots a spray of DNA coated metallic micro-projectiles (micropellets) into the cells.
This device uses high pressure gas to propel the DNA coated projectiles. When pressure of cylinder exceeds the bursting point of plastic disc, it gets ruptured. Helium shock waves propel the plastic micro-carrier containing DNA coated micro-pellets. The transformed cells are regenerated onto nutrient medium and, thereafter analysed for expression of foreign DNA.
4. Gene Bank or Genomic Library:
Gene bank or genomic library is a complete collection of cloned DNA fragments which comprises of the entire genome of an organisms.
Gene bank is constructed by a shotgun experiment method where whole genome of a cell is cloned in the form of random and unidentified clones. The steps of constructing gene bank of an organism section-plaque hybridization technique.
Table 11.4 : Some human peptides and proteins synthesized by gene technology.
