12a Bilegdemberel

Gene Technology

Gene technology is the manipulation of genes in living organisms. Genes from one organism may be inserted into another. This may be done within the same species ( for example in gene therapy ) or genes may be transferred from one species to another.

What is gene technology

Insulin is a small protein. It is hormone secreted by β cells in the islets of Langerhans in the pancreas in response to raised blood glucose concentration. In type I one diabetes, no or insufficient insulin is secreted, and the person has to inject insulin. This used to be obtained from animals such as pigs. Today, almost all insulin used in this way is obtained from genetically modified bacteria.

Gene technology for insulin production

The amino acid sequence of insulin was already known. From this, the probable base sequence of the gene that codes for it, and of the mRNA transcribed from the gene could be worked out.

Identifying the insulin gene

Messenger RNA was extracted from β cells. These cells express the gene for insulin, so much of this mRNA had been transcribed from this gene. The appropriate mRNA was then incubated with the enzyme reverse transcriptase, which built single-stranded cDNA molecules against it. These were then converted to double-stranded DNA- the insulin gene.

Making the human insulin gene

Some extra single-stranded DNA was then added to each end of the DNA molecules. These are called sticky ends. Because they are single stranded, they are able to form hydrogen bonds with other single-stranded DNA, enabling DNA molecules to join up with one another. This is important in a later stage of the process.

Making the human insulin gene

Multiple copies of the DNA were then made using DNA polymerase. This can be done using the polymerase chain reaction, or PCR. A small amount of DNA is incubated with DNA polymerase in a repeated sequence of changing temperatures, enabling a huge number of copies to be made in a relatively short period of time.

Cloning the DNA

A plasmid is a small, circular DNA molecule found in many bacteria. A plasmid was cut open using a restriction enzyme. The restriction enzymes make a stepped cut across the DNA molecule, leaving single stranded regions.

The cut plasmid and the insulin gene were then mixed together, along with the enzyme DNA ligase. Complementary base pairing took place between the sticky ends of the cut plasmids. DNA ligase then joined up the sugar-phosphate

Inserting the DNA into a plasmid vector

Backbones of the DNA strands. This resulted in closed plasmids containing the insulin gene.

Genes conferring resistance to an antibiotic were also introduced into the plasmids, next to the insulin gene.

Not all of the plasmids took up the gene. Some just closed back up again without it.

Inserting the DNA into a plasmid vector

The plasmids were mixed with a culture of the bacterium Escherichia coli. About 1% of them took up the plasmids containing the insulin gene.

Inserting the plasmid vector into a bacterium

Antibiotics were then added to the culture of E. Coli bacteria. The only ones that survived were the ones that had succcessfully taken up the plasmids containing the antibiotic resistance gene. Most of these plasmids would also have contained the insulin gene. Most of the surviving E. Coli bacteria were therefore ones that now contained the human insulin gene.

Identifying the genetically modified bacteria

The bacteria were then grown in fermenters, where they were provided with nutrients and oxygen to allow them to form large populations. Reproduction is asexual, so all the bacteria were genetically identical.

This is now done on a large scale. The bacteria synthesise and secrete insulin, which is harvested from the fermenters and purified before sale.

Cloning the bacteria and harvesting the insulin

It is identical to human insulin, because it is made following the genetic code on the human insulin gene. Insulin obtained from the pancreas of an animal is slightly different, and therefore may have different effects when used to treat diabetes in humans.

Large quantities of insulin can be made continuously using E. Coli, and this can be done under controlled conditions.

Advantages of insulin produced by gene technology

Only small quantities of insulin can be obtained from the pancreas of an animal, and it is not easy to purify the insulin to produce standard product that is safe for medicinal use.

Many religions and cultures, and also many individuals, are uncomfortable with idea of harvesting insulin from a dead animal for use in humans.

Advantages of insulin produced by gene technology

In bacteria, each gene is associated with a region of DNA called a promoter. The enzyme RNA polymerase must bind to the promoter before it can begin transcribing DNA to produce mRNA.

It is therefore important to ensure that there is a promoter associated with the human insulin gene when it is inserted into E.Coli

Promoters

The antibiotic resistance genes added to the plasmids along with the human insulin gene act as markers. They make it possible to identify the bacteria that have taken up the gene.

There is a concern that using antibiotic resistance genes as markers could increase the likelihood of the development of populations of harmful bacteria that are resistant to antibiotics. Today, most common markers used are genes that code for the production of fluorescent green protein. The gene for this protein can be inserted along with the desired gene. Cells that fluoresce green are therefore likely to have taken up the desired gene.

Markers

Electrophoresis is way of separating strands of DNA of different lenghts.

Cut DNA by restriction enzymes

Place on agarose gel

Apply current

Fragments travel toward anode

Short fragments travel further

Visualise DNA with UV light

Electrophoresis

Cystic Fibrosis (CF) is a genetic condition resulting from a mutation in a gene that codes for a transporter protein called CFTR. CFTR protein actively transport Cl- ions out of cells. When CFTR protein is not working, high concentration of chloride ion builds up inside the cell. It is because chloride ions are actively transported into the cell but not out of the cell. Therefore water moves into the cell resulting in thick and sticky mucus.

Cystic Fibrosis

The abnormality thick mucus collects in the lungs, interfering with gas exchange and increasing the chance of bacterial infections.

The pancreatic duct may also become blocked with sticky mucus, interfering with digestion in the small intestine.

Reproductive passages , such as vas deferens, may become blocked, making a person sterile.

Cystic Fibrosis

Cystic fibrosis

Finding out the genes that a person has is called genetic screening. Genetic screening can be used:

To identify people who are carriers, that is who have a copy of a harmful recessive allele, such as the cystic fibrosis in the family could therefore find out if they are both hererozygous and therefore might have a child with cystic fibrosiss.

In pre implantation genetic diagnosis, to check the genes of an embryo produced in vitro before it is placed in the mother’s uterus.

Screening for genetic conditions

For prenatal testing, that is checking the genes of an embryo or fetus in the uterus;

To identify people who will develop a genetic condition later in life

To identify people with alleles that put them at risk of developing other diseases.

Screening for genetic conditions

Thank you !