BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Vectors

Vectors are DNA molecules used to transfer genes from a donor Organism to a recipient organism, as well as to clone nucleotide sequences.

An ideal vector molecule should possess the following properties:

- persist in the recipient Cell population for a prolonged period;

- contain biochemical or genetic markers to facilitate the detection of the vector within Cells;

- The Structure of the vector molecule must allow the insertion of a foreign nucleotide sequence without disrupting its functional integrity.

Plasmids, cosmids, viral Chromosomes, bacterial artificial chromosomes, Bacteriophages, Yeasts, and mammalian artificial chromosomes are used as vectors (Table 1.1).

Class="center">Table 1.1

Comparative characteristics of various vectors for DNA Cloning

Plasmid Vectors are most commonly used in Introduction/32.html">Genetic Engineering. Plasmids are extrachromosomal, double-stranded, circular DNA molecules that replicate autonomously. Within The Cell, plasmids function like genes, directing the synthesis of new Proteins.

The first plasmid was discovered by J.J. Kelly, H.O. Smith, and K. Wilcox in 1968. American biochemist Stanley Cohen suggested that plasmids could be used for Gene transfer. Experiments were conducted with plasmids from various bacterial species. The scientists created recombinant plasmids from DNA fragments of Staphylococcus aureus (conferring penicillin resistance) and Escherichia coli (conferring tetracycline resistance). The hybrid plasmids were introduced into E. coli cells, and the resulting strain was resistant to both penicillin and tetracycline. This experiment proved that Genetic information can be transferred between different species of organisms.

Plasmids are found in virtually all Bacteria. The main bacterial chromosome contains 1000–5000 genes. Plasmids contain less than 5% of genetic information (50–250 genes). Some carry genes responsible for their transfer from one cell to another (F-plasmids), others contain Antibiotic Resistance genes (R-plasmids), or genes encoding the degradation of xenobiotics (degradation plasmids). Plasmid sizes can range from less than 1 to over 500 kbp.

Some plasmids are present in cells at 10–100 copies and are termed high-copy plasmids. Low-copy plasmids are found in cells in quantities of 1–4 copies. Plasmid DNA accounts for 0.1–5% of the total cellular DNA content.

Plasmids of the pBR series are used as a basis for constructing plasmid vectors. These plasmids were derived from a natural plasmid that confers colicin resistance to E. coli cells. A drawback of plasmid vectors is their small cloning capacity.

Bacteriophage λ chromosome-based vectors are used when large DNA fragments need to be inserted. The λ phage DNA molecule is about 45 kbp long, and it contains a 15 kbp region in the middle that can be replaced with any DNA fragment. Vectors based on the bacteriophage λ chromosome can accommodate DNA fragments 15–25 kbp in length.

Viral vectors are used in experimental Human Gene Therapy. A disadvantage of bacteriophage λ chromosome-based vectors is that the genetic material integrates into random sites of the DNA molecule, making the gene transfer process difficult to control.

Cosmids are small plasmids into which bacteriophage λ DNA cos-sites have been introduced in vitro. Cos-sites are located 35–45 kbp apart; the entire phage genome situated between the cos-sites can be replaced with a foreign DNA fragment of equivalent length. Cosmids can incorporate up to 40 kbp of foreign DNA while actively amplifying within E. coli cells.

Phasmids are vector DNA molecules that contain genetic elements of both plasmids and bacteriophage chromosomes.

Artificial chromosomes are double-stranded DNA molecules with terminal telomeres, a centromere, and binding sites for DNA polymerase. To introduce large DNA fragments into differentiated and embryonic cells, bacterial artificial chromosomes (BAC), bacteriophage artificial chromosomes (PAC), yeast artificial chromosomes (YAC), mammalian artificial chromosomes (MAC), and human artificial chromosomes (HAC) have been developed.

Bacterial artificial chromosomes, developed in 1992, are the most widely used. They are based on the E. coli F-plasmid, bacterial telomeres, and a centromere. These structures allow the introduction of large DNA fragments up to 300 kbp in length into the cell. Bacterial artificial chromosomes are relatively stable within the bacterial genome, replicating during bacterial growth and division.

A. Murray and J. Szostak (1983) and D. Burke (1987) pioneered the creation of the first yeast-based artificial chromosomes. Such artificial chromosomes can accommodate an even larger volume of genetic material — DNA fragments up to 1 Mbp in length. Their drawback is lower stability compared to bacterial artificial chromosomes.

Animal and human artificial chromosomes make it possible to introduce entire genetic loci and achieve expression of the corresponding genes in cell culture or Transgenic Animals. The first human artificial chromosomes were created in 1997. Artificial chromosomes do not integrate into the host cell genome; instead, they function within The Cell as autonomous minichromosomes, directing Gene Expression. Artificial chromosomes are introduced into cells via electroporation, where electrical pulses create pores in The cell membrane through which the artificial chromosomes enter the cell.