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In Vivo Cloning

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Biology

What is in vivo cloning? How is it different from the in vitro method, and what steps does it follow?

Gene cloning

Once we have obtained a DNA fragment, it needs to be amplified for medical or commercial applications. This is done by cloning the desired gene by creating many copies of it. There are two ways to clone a gene: in vivo and in vitro.

In vivo

Latin for ‘within the living.’ It involves transferring the gene into a host cell using a vector.

In vitro

Latin for ‘in the glass.’ It involves using the polymerase chain reaction (PCR) in the lab.

Sticky ends

A sticky end is the endpoint of a DNA double helix where a few unpaired nucleotides from one strand protrude beyond the other.

As mentioned in the Gene Technology explanation, one way to create DNA fragments is by using restriction endonuclease enzymes which cut the DNA at specific locations called restriction sites.

These restriction sites have a palindromic sequence. This means that they have the same sequence backwards and forwards while being complementary to each other. There are various types of restriction endonucleases. Some of them cut the DNA at different positions on the two DNA strands which are a few bases apart. This leads to the formation of staggered ends at which out of the double DNA strands, one is a few bases longer with unpaired nucleotides.

It is important to note that if multiple DNA fragments with the same restriction endonuclease are created, the staggered ends of the fragments would contain unpaired bases that are complementary to other fragments. Therefore, the single-stranded part of any of the DNA fragments can join the single-stranded part of any other DNA fragment. Hence causing the ends to be sticky.

The complementary bases can pair up forming hydrogen bonds but they are weak. For permanent adhesion of the DNA fragments, DNA ligase needs to be used to create phosphodiesterase bonds between the phosphate and sugar groups of the adjacent DNA nucleotides.

In vivo cloning

In both eukaryotic and prokaryotic systems, RNA polymerase initially binds to the regulatory regions of the DNA. This is called the promoter and is adjacent to the gene. Transcription factors also bind to the promoter regions and assist recruitment of the RNA polymerase to the promoter. Without the promoter region, the DNA cannot be transcribed. Therefore, before inserting the DNA fragments into a host cell, we need to add a promoter region to the start of the gene.

Genes in the cells also contain a terminator region at which the RNA polymerase dissociates from the DNA and ends the transcription. Hence, we need to add a terminator region to the end of the gene.

Insertion into a vector

So far we have learned how recombinant DNA is produced. To introduce the recombinant DNA into a cell, we first need to insert it into a vector. A vector is any carrier, usually a virus or plasmid, that is used to transport a desired piece of DNA into a host cell.

Plasmids

Small circular DNA molecules found in bacteria. They usually contain genes for antibiotic resistance.

We use the same restriction endonucleases used for creating the DNA fragments to cut open the plasmid. This is due to the formation of complementary sticky ends in the plasmid and the desired DNA fragment which facilitates the incorporation of the DNA fragment into the plasmid. When the sticky ends of the plasmids and the DNA fragments are lined up, DNA ligase is used to join the sugar-phosphate backbone of the strands and make their adhesion permanent.

At the end of this process, plasmids would be containing recombinant DNA.

Transformation of host cells with the vector

Transformation involves the introduction of recombinant DNA into the host cells.

In the case of bacterial cells, transformation is the reintroduction of recombinant plasmids into bacterial cells. This process requires the presence of calcium ions and changes in the temperature to make the plasma membrane permeable to the passage of plasmids.

The process of transformation often does not have a high yield. This is for three reasons:

  1. Not all bacterial cells successfully uptake the recombinant plasmids.
  2. Some of the plasmids may have been closed up without the incorporation of the desired gene.
  3. The sticky ends of some of the desired DNA fragments may have also paired up with each other without being incorporated into a plasmid.

Identification of cells with the plasmid

Since the transformation process does not have a high yield, it is important to have a strategy for selecting the host cells that have taken up the plasmids. This selection is achieved by using antibiotics. Plasmids usually contain the genes that confer resistance to antibiotics. These genes often code for enzymes that can break down the antibiotic molecules to prevent the destruction of bacterial cells.

Some plasmids may often contain different genes that confer resistance to two different antibiotics. For example, R-plasmid contains genes for resistance against ampicillin and tetracycline.

All the bacteria are grown in a medium that contains an antibiotic to use antibiotic resistance to select for bacteria that have taken up plasmids. Bacterial cells that have taken up the plasmid are resistant to the antibiotic and will survive. Meanwhile, other bacteria that have failed to take up the plasmid remain susceptible to the antibiotic and will die.

Not all the cells that have acquired resistance to the antibiotic contain the desired gene. This is because some plasmids may have closed up without incorporating the gene. Therefore, there need to be some strategies for selecting the cells that have taken up the recombinant plasmids.

Selection of cells that have acquired the desired gene

Scientists commonly use marker genes for identifying the cells that have been successfully transformed with the desired gene since they are located on the same DNA fragment that the desired gene is.

We normally use three different types of marker genes for this purpose:

Antibiotic resistance genes

Plasmids may often contain different genes that confer resistance to two different antibiotics.

For example, R-plasmid contains genes for resistance against ampicillin and tetracycline. These genes often code for enzymes that are able to break down the antibiotic molecules to prevent the destruction of bacterial cells.

The restriction endonucleases used to cut open the plasmid are designed to have their restriction sites in the middle of an antibiotic resistance gene. Therefore, when the desired DNA fragment is successfully incorporated into the plasmid, it also results in the bacteria no longer being resistant to that antibiotic. For instance, if the restriction enzyme cuts the R-plasmid at the tetracycline resistance gene, the recombinant plasmid would no longer contain a functional tetracycline resistance gene. So, if the cells are grown on a medium that contains tetracycline, the cells with recombinant DNA will die.

This causes a problem since the cells that we are interested in would be dead. To tackle this problem, we use the replica plating technique.

Fluorescent markers

Fluorescent marker genes code for proteins that fluoresce and glow when they absorb light. One example of these is the green fluorescence protein (GFP) which was first identified in jellyfish.

When GFP is added to the DNA fragment that contains the desired gene, the cells that have successfully acquired the recombinant plasmid would then express the GFP. They would then appear green. Since there is no need for replica plating, fluorescent markers make the selection process easier.

Enzyme markers

Similar to GFP, we can use enzymes such as lactate to select recombinant cells. The lactase enzyme needs to be attached to the DNA fragments with the desired gene. The cells that have acquired the recombinant plasmid would then produce lactate. Lactate can turn a colourless substrate blue. Therefore, in presence of the recombinant cells, the substrate would turn blue.

In Vivo Cloning - Key takeaways

  • In vivo is Latin for ‘within the living.’ It involves transferring the gene into a host cell using a vector.
  • Before inserting the DNA fragments into a host cell, a promoter and a terminator region need to be added to the start and the end of the gene, respectively.
  • The transformation process requires the presence of calcium ions and changes in the temperature to make the plasma membrane permeable to the passage of plasmids.
  • There are three methods for selecting the recombinant cells that have acquired the desired gene. These include:
    • Antibiotic resistance genes
    • Fluorescent markers
    • Enzyme markers

In Vivo Cloning

In vivo is Latin for ‘within the living.’ It involves transferring the gene into a host cell using a vector and using the host cell for amplifying the gene.

1. Selecting the host organism and the vector.

2. Processing the vector (plasmid) with restriction enzymes.

3. Processing the desired DNA fragment with restriction enzymes.

4. Formation of recombinant DNA (recombinant plasmid.)

5. Transformation of the host cell with the recombinant DNA.

6. Selection of the transformed recombinant cells.

It is a technique of extracting a certain DNA sequence in order to make many copies of it.

Gene cloning is used for amplifying and making multiple copies of a gene of interest. It can be done either in vivo or in vitro.

In vivo means ‘within the living,’ hence an in vivo test is one that is conducted in the body of a living subject.

Final In Vivo Cloning Quiz

Question

What is in vivo gene cloning?

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Answer

In vivo is Latin for ‘within the living.’ It involves transferring the gene into a host cell using a vector to make many copies of the gene.

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Question

What is in vitro gene cloning?

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Answer

In vitro is Latin for ‘in the glass.’ It involves using the polymerase chain reaction (PCR) in the lab.

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Question

What is the name of the site where the restriction endonucleases cut the DNA?


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Answer

Restriction sites

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Question

What is the characteristic of restriction sites?


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Answer

They have a palindromic sequence.

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Question

What are sticky ends?


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Answer

The single-stranded part of any of the DNA fragments can join the single-stranded part of any other DNA fragment hence causing the ends to be sticky.

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Question

What is the promoter?


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Answer

In both eukaryotic and prokaryotic systems, RNA polymerase initially binds to regulatory regions of the DNA called the promoter which is adjacent to the gene.

Show question

Question

What is the terminator region?


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Answer

The region of DNA after the gene where the RNA polymerase dissociates.

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Question

What is a vector?


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Answer

A vector is any carrier, usually a virus or plasmid, that is used to transport a desired piece of DNA into a host cell.

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Question

What is the transformation of host cells and what conditions does it require?


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Answer

Transformation involves the introduction of recombinant DNA into the host cells. It requires calcium ions and a change in temperature.

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Question

Why does transformation have a low yield?


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Answer

1. Not all bacterial cells successfully uptake the recombinant plasmids.

2. Some of the plasmids may have been closed up without the incorporation of the desired gene.

3. The sticky ends of some of the desired DNA fragments may have also paired up with each other without being incorporated into a plasmid.

Show question

Question

The R-plasmid contains genes for resistance against _________ and _________.


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Answer

Ampicillin and tetracycline

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Question

What are the three common types of marker genes?


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Answer

Antibiotic resistance genes, fluorescence markers, and enzyme markers.

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Question

What is GFP?


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Answer

Green fluorescent protein is a fluorescence marker first identified in jellyfish.

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Question

What is the name of the enzyme that permanently joins two DNA fragments?


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Answer

DNA ligase

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Question

What is the characteristic of the lactase marker?


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Answer

Lactase converts a colourless substrate to blue.

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