Question

1. Compare calcium chloride transformation, electroporation and transfection. State the benefits and drawbacks of each method and describe the situations in which each method may be used.
2. What are the challenges of using biolistics for genetically modified organisms? Give one case study on the use of biolistics.
3. Explain the use of Agrobacterium tumerfaciens in genetically modifying crops?

Answer 1

Calcium chloride transformation

Competent cells are bacterial cells that can accept extra-chromosomal DNA or plasmids (naked DNA) from the environment. The generation of competent cells may occur through two methods: natural competence and artificial competence. Natural competence is the genetic ability of a bacterium to receive environmental DNA under natural or in vitro conditions. Bacteria can also be made competent artificially by chemical treatment and heat shock to make them transiently permeable to DNA. This process of making bacterial cells competent by making small holes in the bacterial cell is done using calcium chloride. The calcium chloride transformation technique is the most efficient technique among competent cell preparation protocols. It increases the bacterial cell’s ability to incorporate plasmid DNA, facilitating genetic transformation. The addition of calcium chloride to the cell suspension allows the binding of plasmid DNA to LPS. Thus, both the negatively charged DNA backbone and LPS come together and when heat shock is provided, plasmid DNA passes into the bacterial cell.

Advantage and disadvantages

The cost- efficiency and simplicity of the method allows its use in many different cultures, this method can accommodate a high concentration of DNA. One of the drawbacks is that the Calcium chloride method has only 10% efficiency. Also, sometimes the calcium chloride can disrupt the cell membrane and cause cell death. Thus, it is not suitable for protocols that require a long incubation period.

Electroporation.

Electroporation is a physical transfection method that uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells. It is a highly efficient strategy for the introduction of foreign nucleic acids into many cell types, including bacteria and mammalian cells. Electroporation is based on a simple process. Host cells and selected molecules are suspended in a conductive solution, and an electrical circuit is closed around the mixture. An electrical pulse at an optimized voltage and only lasting a few microseconds to a millisecond is discharged through the cell suspension. This disturbs the phospholipid bilayer of the membrane and results in the formation of temporary pores. The electric potential across the cell membrane simultaneously rises to allow charged molecules like DNA to be driven across the membrane through the pores in a manner similar to electrophoresis

Advantage and disadvantages

The main advantage of electroporation is its applicability for transient and stable transfection of all cell types. Furthermore, because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined. The major drawback of electroporation is substantial cell death caused by high voltage pulses and only partially successful membrane repair, requiring the use of greater quantities of cells compared to chemical transfection methods

This process is used because It can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps.

Transfection

Transfection is the process of delivering nucleic acids and small proteins into eukaryotic cells. This can be achieved by using chemical transfection reagents, electroporation (gene electrotransfer), viral transduction, and several other less common methods. The ultimate goal of transfection is to deliver nucleic acids into cells so as to investigate gene function. This can be accomplished by the expression of exogenous genes or through knockdown of endogenous genes. Manipulation of gene expression is a core technique in research areas such as drug development, cancer research, gene therapy, and tissue engineering.  There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation); others rely on chemical materials or biological particles (viruses) that are used as carriers.

Advantages and disadvantages

The amount of DNA required for transfection is much lesser than the other procedures. The major drawback is its efficiency. This method is used when the gene of interest is in high quantity.

Biolistics methods

Biolistics, or particle bombardment, is a common method for nuclear plant transformation, and for transformations that require DNA to be delivered to chloroplasts and mitochondria. Biolistics is based on the direct delivery of DNA into plant cells using gold or tungsten particles.⁸⁰ The gold or tungsten particles are coated with DNA which is shot at plant tissue at high velocity and can become lodged inside plant cells. Once inside the cell, the DNA elutes off the particles and becomes transiently expressed or stably integrates into the host genome.

Disadvantages include: (i) messy integration patterns, (ii) relatively low throughput, (iii) high input cost, and (iv) the cellular target cannot be controlled (i.e., cytoplasm, nucleus, mitochondria, or plastid).

Case study

Because particle bombardment can transfer genes into freshly excised tissues, making them immediately available for transplantation, it is highly competitive with ex vivo gene transfer, the most common approach for gene therapy. Several laboratories have shown that mammary gland, liver, kidney, and skin explant tissues can be effectively transfected in organ culture via particle bombardment. In the mammary organoid system, the Accell method was 5- to 100-fold more effective than three other commonly used DNA transfer methods. Particle bombardment can be used on cell explants in many forms, such as tissue slices, cubes, clumps, small cell aggregates, or single cells. Suspensions of primary leukocytes–lymphocytes, macrophages, and splenocytes–were also transfected. New opportunities for gene therapy are hence created by the versatility of the particle bombardment technique.

A wide spectrum of mammalian cell lines in long-term culture was also transfected by the particle method, including those derived from epithelial, fibroblast, endothelial, and lymphocyte cells. Stable gene transfer into target cell chromosomes by the bombardment technique was observed at a frequency of ∼10–⁻⁴, similar to that of other direct gene transfer methods

Agrobacterium-Mediated Gene Transfer

The introduction of additional DNA into plant cells or tissues is termed as plant transformation. A DNA of any source can be used for plant transformation. This transformation process has become a very important technique for both scientific and commercial purposes. These transgenic plants, which have a new set of DNA, aid in the understanding of plant gene regulation and also for identification and evaluation of useful traits in plants. Plant transformation is used for the introduction of these useful traits into other plants. Agrobacterium tumefaciens is the most efficient used vector to plant transformation. It has the advantage of evolved crown gall-inducing mechanisms of DNA transfer present in the common soil pathogen. The production of transgenic plants involves two major processes. The first step is the introduction of new genetic material into plant cells, a process known as transformation. The second step uses tissue culture techniques for proliferation and to regenerate the transformed cells into transgenic plants. Of various methods used for plant transformation, transformation mediated by Agrobacterium tumefaciens is the most commonly used method.