Genetic Engineering

Genetic engineering is altering the genes in a living organism to produce a genetically modified organism with a new genotype. Recombinant DNA (rDNA) technology is a technique by which the gene (or segment of DNA) is excised from one organism and introduced into another organism. The process of introducing the foreign gene into another organism (or vector) is also called cloning. rDNA technology is a part of vast field of genetic engineering but some authors consider them synonymously. Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells.

Genetic engineering is altering the genes in a living organism to produce a genetically modified organism with a new genotype. Recombinant DNA (rDNA) technology is a technique by which the gene (or segment of DNA) is excised from one organism and introduced into another organism. The process of introducing the foreign gene into another organism (or vector) is also called cloning. rDNA technology is a part of vast field of genetic engineering but some authors consider them synonymously. Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells.

The genes are identified by various methods, these include
Polysome precipitation
Prediction based on DNA sequence
Alignment with known mRNAs
Homology to known genes
Identification of Start and Stop codons
Finding a gene by using a functional assay
Find a gene by where it is located
Once identified, it is convenient to maintain a gene library.

A gene library is a population of organisms, each of which carries a DNA molecule that was inserted into a cloning vector. Ideally, all of the cloned DNA molecules represent the entire genome of the organism. A gene library is also called gene bank. This term also represents the collection of all of the vector molecules, each carrying piece of the chromosomal DNA of the organism, prior to the insertion of these molecules into the population of the host cell. Since there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries to catalogue the organism's DNA and then select the gene of interest. A cDNA library consists of sum total of all actively transcribed genes of a tissue, inserted into a population of bacterial cells. A total mRNA preparation is reverse-transcribed and inserted into plasmids all at once so that every possible cDNA sequence will be carried by at least one bacterium in the culture.

Isolation of the gene (DNA sequence)
The technique involved in recombinant DNA technology is to slice (cut) the desired DNA segment and introduce it into a vector (e.g., plasmid). This is achieved using a specific bacterial enzyme called restriction enzymes or restriction endonucleases. Will Porter and John Darms received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases. These enzymes function as endonuclease, which can cleave a DNA sequence at a specific site. These enzymes are named with three letters based on the species where it was isolated. For example EcoRI is isolated from E. coli.

Each restriction enzymes cleaves DNA strand at a specific site called recognition sequence or restriction site. For example, Eco RI recognizes the sequence GAATTC and cleaves it between G and A (G↓A).
Enzyme Source Target sequence

BamHI Bacillus amyloliquefaciens G↓GATCC
EcoRI Escherichia coli G↓AATTC
HaeIII Haemophilus aegyptius GG↓CC
HhaI Haemophilus hemolyticus GGG↓C
HindIII Haemophilus influenzae A↓AGCTT
HpaII Haemophilus parainfluenzae C↓CGG
KpnI Klebsiella pneumoniae GGTAC↓C
PvuII Proteus vulgaris CAG↓CTG
TaqI Thermus aquaticus T↓CGA

Sometimes, the restriction sequence occurs on both the strands but in reverse direction. Such a segment of DNA with identical sequences but opposite in direction is called a palindrome. A palindrome site is a sequence of base pairs in double stranded DNA that reads the same backwards and forward across the double strand.

A palindrome



When a restriction enzyme acts on palindrome, it cleaves both the strands of DNA molecule. While some enzymes cut the two strands symmetrically, others cut them asymmetrically. AluI, EcoRV and HaeIII generates blunt ends when they act on their restriction sites. Only those enzymes that cut the DNA asymmetrically are useful in rDNA technology. When such enzymes cleave DNA, they leave single stranded “sticky ends” on both strands. Same restriction enzymes are used to cleave the DNA molecule to be transferred and the vector. The circular structure of the plasmid is broken by the restriction enzyme, this process leaves a “sticky end” at either strand. The strand of DNA to be transferred must have two restriction sites; one on either side of the DNA segment of interest. When it is acted upon by restriction enzyme, it generates two sticky ends, one at either side of the segment. Since these sticky ends are generated by the same enzyme, they are complementary and hence are cohesive.

click to view the picture

Vector
Bacterial plasmid is the most commonly used vector. Plasmids used in genetic engineering are said to be under relaxed control, their replication is totally independent of chromosomal replication. These plasmids may be present in copies of 10-700 per cell.

The most popular plasmid is pUC18. Under certain culture conditions, plasmids can be induced to replicate to produce multiple copies within a single cell. Bacterial plasmids can not accept DNA strands larger than 5000 base pairs, hence they are restricted to cloning DNA ≤5000 base pairs. Some plasmids can carry DNA segments that are 10Kb long.

Specially developed bacteriophage lambda chromosome can incorporate up to 15-16 kilobases of DNA segment. A central one-third of its genome is normally not required for phage infection and therefore can be replaced by foreign DNA. The chimeric phage DNA can be introduced into the host cells by infecting them with phages.

Cosmids are recombinant vectors that combine features of both plasmids and bacteriophage chromosome. It can accommodate DNA segments up to 50 kilobases.
cos site is segment of DNA, which is 14 base pair long sequence and is located at either ends of phage chromosome.These ends have be separated by

36-51 kilobases of DNA strand. Only those segments of DNA that have two cos sides at either end and are separated by 36-51 kb of DNA are packaged into the phage capsid. If two cos sites are placed 36-51 Kb apart on a functional plasmid vector, it becomes a cosmid. Since cosmids have no phage DNA, upon introduction into a host cell via phage infection, they reproduce as plasmids.

Yeast artificial chromosome (YAC) is a specially constructed yeast chromosome that can incorporate DNA strands up to 1 million base pairs. YACs are liner DNA segments that have all the information required for replication in a yeast cell. Several hundred kb of foreign DNA can be cloned into YACs.

The sticky ends are generated by the same enzyme on vector as well as the target DNA are complementary and hence are cohesive. The sticky ends of the cleaved DNA segment cohere with those of the vector, thus the cut DNA sequence can now be introduced into the plasmid. The cut ends are joined by DNA ligase enzyme and the introduced gene becomes a part of the plasmid. Ligase is an enzyme that covalently joins the sugar-phosphate backbone of bases together. Ligase will join either "sticky" ends or "blunt" ends, but it is more efficient at closing sticky ends. The process of introducing foreign gene into a vector is called as cloning and the plasmid containing a cloned gene is called chimera.

Illustration of cloning click to enlarge


If the foreign DNA and the cloning vector does not have a common restriction site at the required position, they may still be spliced through the use of terminal deoxynucleotidyl transferase enzyme. This mammalian enzyme adds nucleotides to 3’-terminal OH group of a DNA chain. It is the only known DNA polymerase that does not require a template. Using this enzyme and dTTP, long poly(dT) tails are build up at the 3’ end of DNA sequence to be cloned. The cloning vector is also enzymatically cleaved at a specific site and 3’ ends of the cleavage sites are extended with poly (dA) tails. The complimentary homopolymer tails are annealed and the strands are joined by DNA ligase. Since the foreign DNA lacks any restriction site, it becomes difficult to recover the insert from the vector.

Illustration on using terminal deoxynucleotidyl transferase enzyme Click to enlarge



Another method to over this problem is to use specially designed palindromic “linker” that are appended to either ends of the DNA insert. This linker is a chemically synthesized DNA fragment that has the same restriction site present in the vector. The linkers are attached to inserts by blunt end ligation with T4 ligase. They are then cleaved with appropriate restriction enzyme resulting in generation of sticky ends at either sides of the insert. The sticky ends of the vector and those of target DNA sequence (with linker) cohere. The strands are annealed and ligated by DNA ligase enzyme.

Illustration on using palindromic linker Click to enlarge


The wild λ bacteriophage has a genome of 48.5 kb, of which the central 1/3rd is not essential for infectivity. Genetically engineered λ phage variants contain restriction sites that flank the dispensable central third of genome. This segment may be cleaved by specific restriction enzyme and replaced be a foreign DNA segment of almost same length. The foreign DNA segment is annealed to the nicked phage DNA and ligated. Only those DNA segments that have length similar to the wild phages gets packaged into the heads. Those phages that have incorporated the “chimeric” chromosome become infectious.

Illustration on using λ phage Click to enlarge


Sometimes when human DNA is inserted into bacterial plasmid, it may not get expressed despite the presence of promoter. This is because bacteria RNA polymerase may not recognize promoter of human origin. This problem can be overcome by replacing human promoter region with bacterial promoter upstream of the gene. Such a vector containing bacterial promoter region (that result in expression of foreign gene) is called expression vector.

The DNA sequence that has been inserted into the vector is also called an “insert”.

The chimera is then introduced into its host (e.g.,a bacterium) by various methods. Vectors carrying the genes must be incorporated into the living cells so that they can be expressed or replicated. The cells receiving the vector is called the host cell and once the vector is successfully incorporated into the host cell, the host cell is said to be “transformed”.

Illustration of cloning in bacteria


Foreign DNA cannot be readily sent across the membrane, following are few methods.
Heat shock: The chimera plasmids are placed in a solution containing cold calcium chloride and normal host bacteria. On heating suddenly to 42°C for 2-5 minutes the host bacterial membranes become permeable to plasmid chimeras, which pass into the cell.
Electroporation: The host cells are subjected to a high voltage pulse which temporarily disrupts the membrane and allows the vector to enter the cell.
Viruses: Since viruses have mechanism to infect susceptible cell and replicate themselves, a genetically engineered virus can deliver desired DNA sequence into the target host cell.
Gene gun: Gold particles coated with foreign DNA segments are fired into the host cell.
Microinjection: A cell in held in place with a pipette under a microscope and foreign DNA is injected directly into the nucleus using fine needle.
Liposome: Vectors can be enclosed in a liposome, which are small membrane bound vesicles. The liposomes fuse with the cell membrane (or nuclear membrane) and deliver the DNA into the cytoplasm/nucleus.

Selection of transformed cells
A pUC18 plasmid containing gene (lacZ’) coding for galactosidase activity is inserted with a foreign DNA. The plasmid also codes for ampicillin resistance. Due to the insertion, the gene gets interrupted and the bacterium transformed with this plasmid lacks galactosidase activity. Bacteria lacking this plasmid as well as those transformed by the chimeric plasmid lack galactosidase activity. When grown on medium containing a chromogenic substrate, bacteria containing chimeric plasmid produce colourless colonies.
Bacteria containing plasmid without the insert produce blue colonies and the bacteria not transformed by plasmid also produce colourless colonies. If ampicillin is also incorporated in the medium, bacteria not transformed with plasmid do not produce colonies. Thus, on this medium the colourless colonies indicate bacteria that have received chimeric plasmid.
Other methods to detect successful transfer of DNA include DNA hybridization and PCR.

Gene mapping
It involves determining the locations of genes within specific chromosomes. It is a critical step in the understanding of genetic diseases. There are two types of gene mapping, genetic mapping and physical mapping. Genetic mapping is used to determine the relative position of genes within a chromosome. This is measured by whether or not two genes are "linked". If both genes are inherited together they are considered linked. By determining which genes are linked, the relative positions of genes can be worked out. Physical mapping involves determining the exact position of a specific gene within a chromosome. There are multiple techniques for accomplishing this, including somatic cell hybridization and Fluorescent In Situ Hybridization (FISH).

Applications of Genetic Engineering
Genetic engineering has wide, applications in modem biotechnology. For various industrial processes, this technique may be used in microorganisms as well as with higher organisms. Since microbial cells have a much higher metabolic rate, genes of desired enzymes could be introduced into plasmid of bacteria. Among the medical applications of genetic engineering are the production of hormones, vaccines, interferon, enzymes, antibodies, and vitamins, and in gene therapy for some hereditary diseases.

The bacterial insulin, humulin was prepared by cloning the DNA from chromosome number 11 of human cells in bacteria.

In 1977 Herbert Boyer created E.coli capable of synthesizing somatostatin, the human growth hormone of the brain hypothalamus.

The hormone thymosin alpha-I, as well as Beta-endorphin has been produced by genetically engineered microorganisms.

Subunit vaccines can be prepared by cloning the DNA coding for the antigenic protein present on a pathogen. E.g, Hepatitis B, Foot & mouth disease, malaria etc.
Plants can be made to express antigenically important microbial proteins (edible vaccine).

Weissmann and his associates have produced alpha-interferon by recombinant DNA methods.

The enzyme urokinase, which is used to dissolve blood clots, has been produced by genetically engineered microorganisms.

Chimeric monoclonal antibodies with human Fc region can be made using this technology.

This technology has been applied to treat some of the genetic diseases (gene therapy), but they are mainly in the experimental stages.

AAT (alpha-1 antitrypsin- used in treating emphysema), tissue plasminogen activator, factor VIII, antithrombin, erythropoeitin etc are some of the other proteins produced using this technology.

Transgenic animals with required characteristics can be created by this technique.
A genetically engineered protein called visilizumab has long-lasting clinical benefits for people with the most severe form of ulcerative colitis. Visilizumab specifically binds to CD3 proteins that are expressed on T cells and thus has an inhibiting effect.

Use of recombinant human bone morphogenetic protein, or rhBMP-2, has been approved for certain types of spine fusion surgery. RhBMP-2 is a genetically engineered version of a naturally occurring protein that helps to stimulate bone growth.
Human keratin has been genetically engineered with the aim of incorporating the compound into skin and hair care products.