Gene Delivery Systems: Steps, Types!
Contents
Introduction:
The delivery and expression of foreign genes into the plant cells is one of the most recent and potential areas of plant biotechnology.
It involves the approach of transfer of specific genes in plants through various transformation techniques, thus resulting in the production of GMPs (Genetically Modified Plants). In case of plants, meristem cells, protoplast, pollen or zygotes are used for gene transfer followed by regeneration leading to the formation of transgenic plants.
The gene delivery or gene transfer technology in plants involves the following steps:
i. Selection of target cell, desired gene and suitable vector.
ii. Insertion of desired gene into the vector.
iii. Transfer of this vector (containing desired gene) into the target cell.
The target cell which is now a transformed cell is regenerated and the expression of transferred genes is observed. During the process of delivery of gene, the activity and the efficiency of restriction enzymes and ligation enzymes is the most important factor.
Types of Gene Delivery Systems:
Three main types of gene delivery systems have been in use with varying degrees of success with higher plants. These are:
(a) Agrobacterium Mediated Gene Transfer (AMGT) System:
The bacterium Agrobacterium is known as the Nature’s smallest Genetic Engineer. It is a pathogenic, gram-negative, soil bacterium.
Agrobacterium tumefaciens causes Crown-Gall disease in dicotyledonous plants and contains Ti-plasmid i.e., tumour inducing plasmid. A. rhizogenes causes Hairy-Root disease and contains Ri -plasmid i.e., root inducing plasmid. The vectors based on Ti-plasmid and Ri-plasmid are used in this type of Gene Transfer system.
(b) Gene Transfer Utilizing Plant viruses as Vectors:
Plant viruses may also be used as the vectors for production of transgenic plants. But there is a problem that most plant viruses have RNA (and not DNA) as their genetic material. RNA-plant viruses need more complex manipulations while used as vectors. Successful experiments have been carried out with DNA-plant virus like caulimovirus and Gemini virus.
Cauliflower Mosaic Virus (CaMV) is most utilized member of caulimovirus group. Maize streak virus (MSV) is an important member of Gemini virus group being used successfully in gene transfer experiments. Apart from these DNA-viruses, some RNA-plant viruses are also employed like Tobacco Mosaic virus (TMV).
(c) Direct Gene Transfer System:
It may also be called as DNA mediated Gene Transfer (DMGT) or vector less gene transfer system. This system does not involve the use of any biological agent like Agrobacterium. The DNA is introduced directly into the plant cell by using certain chemical or physical treatments.
Main methods for direct gene transfer are given below:
(i) Electroporation:
Short electric pulses of high voltage are given to the target cells which results in the formation of temporary small pores in the cell membrane, these pores allow the intake of DNA into the protoplast of cells. (Fig. 1).
(ii) Chemical Methods:
Some chemical helpers are also employed for stimulating direct gene delivery. PEG (Poly Ethylene Glycol) is one such chemical stimulant. PEG precipitates the DNA on outer surface of plasma membrane of target cells and this precipitate is then taken in by endocytosis thus resulting in the entry of DNA into the cell. Calcium phosphate is another example of chemical helper for direct gene transfer.
(iii) Micro-injection:
It is the introduction of new DNA into the target cell by injecting it directly into the nucleus or the protoplast. The target cells are immobilized on a solid surface and the micromanipulator is used for microinjecting the DNA into the cell (Fig. 2).
(iv) Particle-Gun Method:
It is also called micro-projectiles or biolistic method or ballistic method. It involves the bombardment with high velocity micro projectiles onto the target cells. The micro projectiles used are 1-3 mm diameter particles of gold or tungsten coated with the DNA to be transferred (Fig. 3).
History of AMGT:
Agrobacterium mediated gene transfer (AMGT) system was historically the first successful transformation system in plants.
The development of Agrobacterium based vectors was a major breakthrough in the field of genetic engineering. It was discovered that the plasmids carried by bacterial plant pathogens i.e., Agrobacterium result into natural gene transfer. Subsequently, it has been considered and treated as most effective natural genetic engineer.
During the course of history a number of workers, on the basis of their observations, realized the potential of this bacterium for gene transfers. Some of those worth mentioning observations were made by Braun, Morel, Chilton, Barton, etc. It was Braun (1947) who first suggested that probably, Agrobacterium induces a tumor-inducing principle into the plant cell genome on infection.
Later on, Chilton (1977) presented that the tumorous crown galls were produced due to the introduction and integration of few bacterial genes into the plant genome. Finally, utilizing all such information about the importance of Agrobacterium in gene transfers, a number of vectors based on it were gradually developed. Since then, this bacterium is being utilized successfully for production of genetically modified plants.
Classification of Agrobacteria:
The bacterial members of genus Agrobacterium are very beneficial and well established medium for gene transformations in plants. They are gram-negative rod-shaped bacteria. They are usually found in soil at the junction of stem and root of the plants. Genus Agrobacterium belongs to the bacterial family Rhizobiaceae.
Earlier it was classified on the basis of their pathogenicity as follows:
In its recent classification, Agrobactenum has been clashed into different group termed as biotypes which are three in number. This recent classification is made on the basis of growth patterns of the bacteria. Bacterial strains of Biotype-1 and Biotype-2 are used for gene transfer systems. Important bacterial strains belonging to Biotype 1 and 2 are A. tumefaaens and A. rhizogenes respectively.
Biology of Agrobacterium:
Agrobactenum is a soil bacterium and is popularly called as the Natural Genetic Engineer It is known that plasmids do not occur naturally in plants. Thus for plant cell transformation the bacterial plasmids are of great importance, and most of them are based on the Ti-plasmid of Agrobacterium tumefaciens. Some of them are also derived from Ri-plasm, d of A. rhizogenes.
A. tumefacierrs is a pathogenic, gram-negative soil bacterium. It invades the plant through a wound and infects many dicotyledonous plant species and causes the crown gall disease Fig 4) Its ability to cause this disease lies in the Ti-plasmid present within the bacterial cell Another related species is A. rhizogenes. It causes the hairy root disease and contains the Ri-plasmid (Root inducing plasmid).
After infection of plant cell with Agrobacterium, a part of T,-plasmid (or Ri-plasmid) is integrated into the plant chromosomal DNA. This property has led to the attention for using Agrobacterium in genetic transformation techniques.
Ti-plasmid:
Ti-plasmid may also be denoted as pTi. Tumour-inducing plasmid i.e. pTi is responsible for crown-gall disease. It is a large ( ~ 200 kb) plasmid present Within’ the A tumefaciens cell. It carries numerous genes involved in the infective process Non-virulent cells of A. tumefaciens lack this pTi.
When A. tumefaciens infects the plant through any wound, a part of Ti-plasmid gets transferred into the plant genome.
This transferred part of pTi is called T-DNA i.e. transferred-DNA This T-DNA contains few genes which are expressed in the plant cells and produce the cancerous properties in the infected cells. Some of its genes also direct the production of unusual chemical compounds called opines which act as nutrition for Agrobacterium cells.
Important functional regions of Ti-plasmid are:
(i) T-DNA: It is responsible for tumour induction, contains oncogenes and genes for opine synthesis and it is transferred into the plant genome.
(ii) Virulence-region or vir-region: It is responsible for virulence as it regulates the transfer of T-DNA into the host plant cell.
(iii) tra-region: It is responsible for the conjugative transfer of plasmid.
(iv) Opine-catabolism region: It regulates the production of enzymes required by Agrobacterium for utilization of opines.
(v) Ori-site It is the origin of replication.
T-DNA:
The T-DNA (transferred-DNA) is a part of Ti-plasmid (or of Ri-plasmid) which gets transferred into the plant nuclear genome on infection by Agrobacterium. It is a DNA segment of size ̴ 23kb having perfect direct repeat sequence on left and right border. These direct repeat border sequences are 24 bp long and are called Left Border and Right Border They play essential role in T-DNA transfer.
T-DNA carries two main regions:
The oncogenic region that contains the genes responsible for induction of tumour and the opine synthesis region responsible for the synthesis of unusual amino acid or sugar derivatives which are collectively called opines. On T-DNA several gene loci are located which control the occurrence and morphology of tumour (Fig. 5).
Two main regions of T-DNA are:
(i) Oncogenic Region:
It is denoted by one-region.
This region mainly consists of three categories of genes:
(a) tmr:
It induces tumour having large number of roots. It is also designated as Rooty locus. It is responsible for cytokinin synthesis.
(b) tms:
This category has two genes which produce tumour with shoots and this represents shooty locus. These two genes are tmsl and tms2 and they encode for auxin synthesis.
(c) tml:
This gene causes large sized tumours, however its actual function is not known so far.
(ii) Opine Synthesis Region:
It is denoted by os-region. This region consists of the genes involved in the biosynthesis of opines. These genes are arranged towards the right border in T-DNA (Fig. 6).
Transfer of T-DNA into Plant Cell:
The T-DNA is transferred from pTi into the plant genome by a definite process (Fig. 7). Firstly, nicking of T-DNA takes place at specific sites present on both border repeats. As a result of these nicks, a single strand of T-DNA is displaced.
This displaced single strand forms a complex with a protein produced by the vir-region. This protein T-DNA complex then gets transported to the plant nucleus. The T-DNA is believed to enter the plant cell nucleus through nuclear pore complex.
During this whole process of transfer of T-DNA into plant genome, the proteins vir D play important role of recognition of T-DNA border sequence and nicking. The vir D operon encodes for the endonuclease enzyme which produce nicks in the bottom strand of T-DNA at each border between the third and fourth nucleotide base.
Firstly nicking occurs in Right Border (RB) and after this nicking, the vir D protein remains attached to the 5′ end of the single stranded (ss) T-DNA.
Now this ss-T-DNA (bottom strand) is displaced and a new strand is synthesized in place of it. Finally another nick is made on left Border (LB) of T-DNA, generating a completely displaced ss-T-DNA having vir D protein attached to its 5′ end. This ss-T-DNA-vir D complex is then coated with vir E protein.
As a consequence, the diameter of this whole complex reduces for ensuring its easier transfer into plant cell. Once this complex is in the plant cell, the proteins vir E and vir D mediate the uptake of ss T-DNA into nucleus. This ss T-DNA soon becomes double stranded (ds) after arriving the plant nucleus. Finally, this ds T-DNA gets integrated randomly into the plant genome through recombination.
Vir-region:
Virulence-region or vir-region is closely linked to T-DNA in the Ti-plasmid. This region contains a few genes which function for the transfer and integration of T-DNA in the plant genome. This region is essential for virulence i.e., for production of tumour, therefore, it is known as virulence or vir-region.
It is approximately 40 Kb long region organized usually into 8 operons (namely vir A, vir B, vir C, vir D, vir E, vir F, vir G, vir H) which together have about 25 genes. The genes of vir-region do not get transferred themselves, but they only help for the transfer of T-DNA. vir A, B, D, and G are absolute requirements for virulence whereas other operons are required for accessory roles like tumour formation.
Vectors for Gene Delivery Based On Ti and Ri-Plasmids:
The unique property of Ti-plasmid (or Ri-plasmid) of transferring their T-DNA into the plant genome allows us to use them for transporting new genes into plant cells.
The only thing to be done for this is to insert the new genes into the T-DNA and after that all will be done by Agrobacterium itself. pTi or pRi cannot be used directly as vectors due to their large size and absence of unique restriction site. So, novel strategies are used for inserting new DNA into the plasmid (pTi or pRi) to construct the plasmid vectors.
Such constructed Agrobacterium-based plasmid vectors may be of following types:
(i) Oncogenic vectors
(ii) Non-oncogenic vectors
(a) Co-integrative type vectors
(b) Binary Vectors.
(i) Oncogenic Vectors:
These are the simplest vectors based on Ti/Ri plasmids. However, they have very limited use because, as the name suggests, they produce tumorous transformed tissues.
Normal mature plants cannot be regenerated from such transformed tumour tissues. One of the most useful oncogenic vectors is pGV3851. Which is constructed by deletion of internal region of nopaline T-DNA and replacing it with pBR322 sequence.
(ii) Non-Oncogenic Vectors:
These are the most widely used vectors as they allow the regeneration of normal plants from the transformed cells. An essential step in designing such vectors is disarming of the plasmid.
A Ti/Ri plasmid from which the oncogenes of T-DNA have been removed (or replaced by other genes) so that it is no longer able to promote cancerous growth of plant cells is called disarmed plasmid. The process of removal of oncogenes from Ti/Ri plasmid is called disarming.
The non-oncogenic vectors may be of the co-integrative type or binary type:
(a) Co-Integrative Type Vector:
It is a Ti-vector which is produced by the integration of an intermediate vector into the disarmed T-DNA.
The co-integration event is mediated through a DNA segment common to the Ti-plasmid and the intermediate vector. This co-integration is achieved within Agrobacterium by homologous recombination. The intermediate vector used here is usually a modified plasmid from E. coli, containing the DNA segment to be transformed (Fig. 8).
In co-integrative vector strategy, the desired gene is inserted into a unique restriction site on the small E. coli plasmid (pBR). This pBR which is now acting as an intermediate vector is then introduced, by conjugation, into A. tumefaciens cell, which already carries a Ti-plasmid.
As pBR and pTi have some sequences common to both, so natural recombination occurs. As a result of this recombination, the new gene gets integrated between the T-DNA borders Agrobactcrium cell. On infection of plant cells with this co-integrated T-DNA, the plant cells get transformed (due to insertion of new gene).
pGV3850 is one of the first co-integrative vector which is generated by the nopaline type Ti-plasmid, pTiC58 which is disarmed by replacing its oncogenes by pBR322 sequences (a small E.coli plasmid). Another co-integrative plasmid system developed is SEV system (split-end vector system).
(b) Binary Vectors:
It represents a class of vectors which possesses the vir-region and disarmed T-DNA on separate Ti-plasmids (Fig. 9). That is, a binary vector possesses a pair of plasmids, of which one plasmid is called mini/micro plasmid and the other is called a helper plasmid.
A mini-plasmid contains the disarmed T-DNA sequence with the gene to be transferred. Helper plasmid is a Ti-plasmid which has functional vir-region and lacks TJDNA. The T-DNA of mini-plasmid is transferred into the plant cell genome by the proteins coded by the genes present in helper plasmid.
This binary vector strategy is actually based on the observation that a T-DNA may also remain functional even if it is not attached to the rest of the Ti-plasmid. A binary vector can replicate both in E. coli and Agrobacterium. Binary vectors are usually represented by ‘Bin’ series, for example—pBin19.
Binary vectors have been prepared not only with Ti-plasmids but also with Ri-plasmids from A. rhizogenes. An example of such binary vector is pARS8 which has been used successfully for the production of transformed plants of tomato.
Main Differences of Co-integrative and Binary Vectors:
1. Unlike co-integrative vectors, binary vectors do not require any homology with the resident Ti-plasmid.
2. Binary vectors have the capability to replicate autonomously in Agrobacterium while co-integrative vectors cannot do so.
3. Binary vectors usually do not allow the transfer of unnecessary sequences into the plant genome while it usually occurs with co-integrative vectors.
Producing Transformed Plants Using Agrobacterium:
(a) Oncogenic vector carrying the engineered pTi is made to infect the wound in mature plant the cells in the resulting crown gall will possess the foreign gene. These cells can then be excised and cultured in proper medium to regenerate transformed plants.
(b) The disarmed Ti-plasmid having the foreign gene integrated into T-DNA is placed in Agrobacterium. The plant cells or tissues are then co-cultivated with these Agrobacterium cells for about 2 days.
As a result, the plant cells or tissues get transformed. Such transformed cells are then transferred to suitable media to regenerate into the complete GMPs. This is called co-cultivation technique. Other transformation techniques using Agrobacterium are leaf-disc infection method and floral disc method.
After the transformation of plant cells with Agrobacterium by using above mentioned techniques, the plant cells (or explant) are transferred to a nutrient medium which allows the Agrobacterium to grow, multiply and transfer their T-DNA to the cells of explant.
These explants are then transferred to a bacteriostatic nutrient medium to kill the bacterial cells. The next step is the selection of transformed cells which is achieved by using a selection medium containing appropriate selection agents like herbicides or antibiotics.
The selection agent is decided according to the selectable marker gene employed for the transformation. As a result of selection, the non-transformed cells do not survive. The transformed cells are then allowed for their regeneration into plantlets by supplying proper nutrient media. The plants so obtained are the genetically modified or transgenic plants.
A schematic representation of whole procedure is given below:
Limitations of Using Agrobacterium-Based Vectors:
It is difficult to obtain desirable results with Agrobacterium based vectors in case of monocotyledonous plants, because in nature, A. tumefaciens and A. rhizogenes do not infect monocots.
This is quite disappointing because most of the important crops belong to the category of monocots like-wheat, rice, barley, maize, etc. However several advanced artificial techniques are being used in this direction to obtain better results.
Marker Genes:
A marker gene may be described as a gene whose expression can be efficiently monitored and can be easily detected. During transformation techniques, marker genes are available in the vectors which are then transferred into the host cells also. In all types of transformation techniques, the isolation of transformed cells or tissues is a necessary step and marker genes provide an aid for this isolation.
Like other transformation techniques the isolation is important in Agrobacterium mediated transformation also.
After the explants are inoculated with engineered Agrobacterium there is a need of selection of transformed cells or screening of transformed cells from the non-transformed ones. Marker genes facilitate this selection and screening of transformed cells and are termed as selectable markers and reporter genes respectively.
So, marker genes are of the following two types:
(a) Selectable Marker Genes:
Some marker genes express certain features which allow the survival of only the transformed cells under a particular condition. Such marker genes are referred to as selectable markers.
Usually the selective conditions employed are toxic levels of a substrate like an antibiotic or a herbicide. The selectable markers used here are the genes conferring resistance against these antibiotics or herbicides. Also, for each marker gene there is one substrate.
General steps involved in the selection process by selectable marker genes are:
i. First of all, the selectable marker gene (which provides resistance to herbicide/antibiotic) is introduced into the vector.
ii. This vector containing selectable marker is then introduced into the host cells which are now called transformed cells.
iii. Such transformed cells are cultured on the culture medium which contains toxic level of substrate used for selection (like herbicide or any antibiotic). .
iv. Those cells which are really transformed survive on the medium due to presence of selectable marker genes while the non-transformed cells die due to toxic
Example:
If the marker gene present in the vector is streptomycin resistant gene (spt), then, only the transformed cells survive in the medium having toxic levels of steptomycin (Fig. 10) The non-transformed cells would die due to the absence of spt gene. In this case, spt is the selectable marker.
Some important selectable marker genes with their substrates are given in the Table 1 below:
(b) Reporter Genes:
An alternate method for isolation of transformed cells is by using reporter genes. In this method, screening is done and not selection. It means that in this method, the samples are taken directly from the lot of regenerated shocks. These samples are then checked for the expression of a marker gene.
Here, this marker gene is termed as reporter gene. In simple words, those marker genes which produce specific phenotype and allow easy screening of the ‘cells having them’ from others ‘not having them’ are termed as the reporter genes or screenable genes or scorable marker genes.
A reporter gene is basically a test gene whose expression produces a phenotype which is quantifiable. Reporter gene is very useful in deciding the success of a gene transfer system and also for testing the gene expressions in plants. A reporter gene system plays a valuable role in setting up the standards for parameters deciding the success of any gene transfer technique.
An ideal reporter gene must possess certain common features like its detection should be highly sensitive, it should be detected by putting a minimal effort, it should be detected quantitatively and above all, it should not be expensive.
Example:
gfp gene codes for a Green Fluorescent Protein. It expresses a unique property of emission of green light on exposure to UV-light. Thus the transformed cells having this gene will emit green light in UV-rays while the non-transformed cells will not. Here this gfp gene is a reporter gene or a scorable marker gene facilitating the screening of transformed cells from others. This gfp gene is isolated from a jellyfish.
Some other commonly used reporter genes are given below:
(a) lux gene:
This gene expresses for enzyme luciferase which produces glow/luminescence in dark. This gene naturally occurs in the glow worms and fire flies.
(b) npt II gene:
It encodes for an enzyme Neomycin phosphotransferase. It can be used both as a selectable marker and as a reporter gene. For its detection, radioactive labelling with 32P and then autoradiography is employed.
(c) gus gene:
This gene encodes for an enzyme P-glucuronidase. It results into a coloured reaction inside the plant cell when treated with an appropriate substrate.
(d) Cat gene:
It results into expression of enzyme Chloramphenicol Acetyl transferase. Its presence is identified in the sample by using the technique of autoradiography.
(e) lac Z gene:
It encodes for enzyme β-galactosidase. For its detection, the transformed cells are cultured in a medium containing X-gal. Those cultures which produce colour, contain this gene and those, where no colour appears show the absence of this reporter gene and also of the foreign gene.
(f) nos and ocs genes:
These genes encode for the enzymes which help in synthesis of opines. Gene nos encodes for nopaline synthase and gene ocs for octopine synthase. These genes can be detected in the assay of transformed cells by using electrophoresis.