What are cloning vectors? How are they used in recombinant DNA technology to insert foreign genes into host cells?

Cloning vectors are small, self-replicating DNA molecules that are used to carry foreign DNA fragments into host cells for replication and genetic modification. These vectors serve as vehicles for transferring genes from one organism to another in genetic engineering and biotechnology. A cloning vector must have key features such as an origin of replication (ORI) for self-replication, selectable marker genes (such as antibiotic resistance) for identifying transformed cells, and multiple cloning sites (MCS) with restriction enzyme recognition sequences to facilitate DNA insertion. They vary in size, ranging from 1 kilobases (small plasmids) to over 2000 kilobases (artificial chromosomes), depending on their function and capacity. Cloning vectors are used in gene cloning, recombinant protein production, functional genomics and gene therapy research, making them fundamental tools in biotechnology and genetic engineering.

  1. Plasmid Vectors
  2. Bacteriophage Vectors
  3. Cosmid Vectors
  4. Artificial Chromosome Vectors
    1. Bacterial Artificial Chromosomes (BACs) Vectors 
    2. Yeast Artificial Chromosomes (YACs) Vectors 
  5. Viral vectors
    1. Retroviral Vectors
    2. Adenoviral Vectors
    3. Adeno-Associated Viral (AAV) Vectors
    4. Lentiviral Vectors
Each type has distinct features that make it suitable for specific genetic engineering applications.

How Are Cloning Vectors Used in Recombinant DNA Technology to Insert Foreign Genes into Host Cells?

Recombinant DNA technology involves inserting foreign genes into host cells using cloning vectors, enabling genetic modifications for research, medicine and biotechnology. The process of inserting foreign genes into host cells using cloning vectors involves six major steps:
  1. Isolation of the Desired Gene
  2. Selection of an Appropriate Cloning Vector
  3. Insertion of Foreign DNA into the Cloning Vector
  4. Introduction of the Recombinant Vector into Host Cells
  5. Selection and Screening of Transformed Cells
  6. Expression and Analysis of the Foreign Gene
Each playing a crucial role in ensuring the successful transfer, replication and expression of the desired gene.

1. Isolation of the Desired Gene

The first step in recombinant DNA technology is to isolate the target gene that is to be inserted into the cloning vector. This gene is typically obtained from a donor organism and is responsible for encoding a protein or trait of interest. The isolation process requires precise techniques to extract and purify the desired gene without causing damage.

The gene can be isolated using restriction enzymes, which cut DNA at specific sequences, allowing for the precise extraction of the gene of interest. Another common method is the polymerase chain reaction (PCR), which amplifies the specific gene sequence, producing multiple copies. Once the gene is isolated, it is purified and sometimes modified by adding regulatory sequences like promoters and enhancers to ensure proper expression in the host cell.

For example, if the goal is to produce recombinant insulin, the insulin gene is isolated from human DNA, amplified using PCR and prepared for insertion into a suitable cloning vector.

2. Selection of an Appropriate Cloning Vector

Choosing the right cloning vector is essential for successful gene insertion and expression. The selection depends on several factors, including the size of the gene, the host organism and the intended application.
  • Plasmid Vectors (1–10 kb): Used for cloning small genes in bacteria, such as pBR322 and pUC19.
  • Bacteriophage Vectors (10–23 kb): Used for higher efficiency transformation and genomic library construction. Examples include λgt10 and λgt11.
  • Cosmid Vectors (30–45 kb): Used for cloning larger DNA fragments in bacteria. Examples include pWE15 and SuperCos1.
  • Bacterial Artificial Chromosomes (100–300 kb): Used for large-scale genome sequencing, such as pBAC108L.
  • Yeast Artificial Chromosomes (100 kb–2 Mb): Used for cloning extremely large DNA fragments, such as pYAC4.
  • Viral Vectors (Up to 36 kb): Used for gene therapy and genetic modification in animal cells. Examples include retroviral, adenoviral, lentiviral and adeno-associated viral (AAV) vectors.
Once the appropriate vector is chosen, it must be prepared for gene insertion.

3. Insertion of Foreign DNA into the Cloning Vector

The isolated gene is inserted into the cloning vector using restriction enzymes and DNA ligase. Restriction enzymes cut both the vector and the foreign DNA at specific sites, creating sticky or blunt ends that allow the gene to be inserted precisely.

After cutting, the foreign gene is joined with the vector using DNA ligase, an enzyme that forms stable phosphodiester bonds between the inserted gene and the vector DNA. The resulting molecule is known as recombinant DNA.

In modern techniques, seamless cloning methods such as Gibson Assembly and homologous recombination-based cloning allow efficient insertion without relying on restriction enzymes.

The recombinant vector is then purified and verified using techniques like gel electrophoresis, PCR and DNA sequencing before proceeding to the next step.

4. Introduction of Recombinant Vector into Host Cells (Transformation/Transduction)

Once the recombinant DNA is ready, it must be introduced into host cells using different techniques depending on the vector type and host organism:
  • Transformation (for plasmid vectors): This process involves making bacterial cells competent using heat shock or electroporation, allowing them to take up the recombinant plasmid.
  • Transduction (for bacteriophage and viral vectors): Bacteriophage and viral vectors infect host cells and transfer the recombinant DNA into them.
  • Microinjection and Electroporation (for mammalian and plant cells): Microinjection directly delivers recombinant DNA into cells, while electroporation uses an electric field to make the cell membrane permeable.
Only a small percentage of cells successfully take up the recombinant vector, so selection markers are required to identify transformed cells.

5. Selection and Screening of Transformed Cells

After introducing the recombinant vector into host cells, not all cells will successfully take up the foreign DNA. Therefore, selection and screening are crucial to isolate transformed cells containing the desired gene. This ensures that only genetically modified cells are used for further research and applications.

Selection of Transformed Cells

  • To differentiate transformed from non-transformed cells, selectable marker genes in the cloning vector provide survival advantages. The most common method is antibiotic resistance selection, where vectors carry genes providing resistance to antibiotics like ampicillin or kanamycin. Only transformed cells survive in antibiotic-containing media. Another approach is auxotrophic selection, used in yeast or bacteria, where transformed cells regain the ability to synthesize essential nutrients.

Screening for Recombinant Clones

  • Since some vectors may re-ligate without incorporating the foreign gene, additional screening ensures correct insertion. Blue-white screening uses the lacZ gene, where recombinant colonies appear white, while non-recombinant ones turn blue. PCR screening amplifies the inserted gene, confirming its presence. Restriction digestion analysis and colony hybridization further validate successful cloning. These techniques help confirm that the inserted gene is present and correctly oriented within the vector.

6. Expression and Analysis of Foreign Gene

After confirming the presence of recombinant DNA in transformed cells, the next step is ensuring that the foreign gene is properly expressed and analyzing the resulting protein or RNA. Gene expression refers to the process where the inserted gene is transcribed into mRNA and translated into a functional protein. The success of this step depends on factors such as the promoter, host cell type, and regulatory sequences.

Gene Expression Process:

  • Transcription: The inserted gene is transcribed into mRNA using the host cell’s transcription machinery. A strong promoter ensures efficient RNA production.
  • Translation: The mRNA is translated into protein by ribosomes. Proper codon optimization helps improve protein synthesis in different host systems.
  • Post-Translational Modifications: Some proteins undergo modifications like folding, glycosylation, or phosphorylation, which are essential for their function, especially in eukaryotic cells.

Gene and Protein Analysis

  • mRNA Analysis: Techniques like RT-PCR or Northern blotting check if the gene is being transcribed.
  • Protein Detection: Methods like SDS-PAGE, Western blotting, or ELISA confirm protein production and test its size and activity.
Successful expression and analysis ensure the recombinant gene is functional and can be used in applications like drug production, enzyme manufacturing and research.




Read Also:



Comments

Post a Comment

Popular posts from this blog

UNIT 6 – Transepithelial Transport (Q&A) | MZO-001 MSCZOO | IGNOU

Image
SAQ 1 i) The resting membrane potential is created due to: a) Buildup of the negative ions in the cytosol along the inside of the membrane. b) The separation of positive and negative electrical charges along both sides of the plasma membrane. c) Difference in charge across the membrane of the cell. d) All of the above. Answer: d) All of the above ii) Which of the following factor/s contribute to the resting membrane potential of the cell? a) Unequal distribution of ions in the ECF and cytosol b) Activity of Na⁺/K⁺ ATPases c) Most anions cannot leave the cell d) all of the above Answer: d) All of the above iii) The extracellular fluid (ECF) has high concentration of positively charged sodium ions and negatively charge chloride ions. a) True b) False Answer: a) True iv) Intracellular fluid (ECF) in contact with the cell membrane is positively charged with high concentration of potassium ions, phosphate ions and anionic proteins. a) True b) False Answer: a) True v) Usually, sodium ion...
Read more

UNIT 5 – Biology of Membrane and Transport of lons (Q&A) | MZO-001 MSCZOO | IGNOU

Image
SAQ 1 Fill in the blanks   a) ............. discovered plasma membrane. Answer: Karl Nageli and C. Cramer b) The phospholipid contains .................. charged phosphate group in the hydrophilic part of head. Answer: Negatively c) ................... proposed Sandwich (lipid-protein) model of cell membrane. Answer: Danielli and Davson d) The protein layer present in cell membrane model proposed by Robertson is .................. thick. Answer: 20 A° e) The proteins are aligned properly with the help of ....................... within the lipid bilayer in membrane. Answer: Transmembrane segments SAQ 2 i) Answer in one word: a) Complex integral proteins transmit signals via plasma membrane. Answer: Receptors b) The cellular processes such as movement, growth, division etc. are regulated by this property of membrane. Answer: Fluidity c) No energy is required for transter of substances from high concentration zone to low concentration zone in this proces. Answer: Passive d) Cer...
Read more

What is the role of actin and myosin in muscle contraction?

Image
Actin and myosin are two primary filamentous proteins that play a direct and central role in muscle contraction. These proteins are located inside the sarcomere, which is the basic contractile unit of striated muscle fibers. Their interaction is responsible for producing the force and movement necessary for muscle contraction, based on the  Sliding Filament Theory  proposed independently by Huxley and Niedergerke, and Huxley and Hanson in 1954. Role of Actin Actin forms the  thin filaments  of the sarcomere. It is a polymer of globular actin (G-actin), which forms a helical structure called  filamentous actin (F-actin).  Each actin filament contains binding sites for the heads of myosin. These sites are normally blocked by a regulatory protein called  tropomyosin,  which is held in place by a calcium-sensitive complex known as  troponin.  When a nerve impulse stimulates the muscle, calcium ions are released from the sarcoplasmic reticulu...
Read more

Fluid mosaic model of the plasma membrane

Image
The fluid mosaic model is one of the most widely accepted and foundational concepts that explains the structural organization of the plasma membrane in living cells. It was first proposed by  S.J. Singer and Garth L. Nicolson in 1972  and remains relevant today, though slightly refined with modern findings. Before we move into the components and structure, it is important to understand that this model helps explain how the membrane remains flexible, selectively permeable, and functionally active for processes like transport, cell signaling and communication. Basic Concept of the Fluid Mosaic Model According to the fluid mosaic model, the plasma membrane is viewed as a dynamic, semi-fluid bilayer of lipids with proteins embedded in it, much like boats floating in a sea. The word  "fluid"  refers to the lateral movement of lipids and some proteins within the membrane, while  "mosaic"  refers to the patchwork arrangement of various proteins that float freely o...
Read more

What neurological condition is caused by inhibiting GABA?

When GABA is inhibited in the brain, it leads to a neurological condition called  epilepsy.  This happens because GABA normally controls brain activity by stopping too much nerve signalling. When GABA is not working properly, the brain loses its balance and nerve cells start becoming overactive. This overactivity can lead to  seizures,  which is the main feature of  epilepsy. Seizures  are sudden, uncontrolled electrical disturbances in the brain that can cause changes in behavior, movements, feelings, or consciousness. They may last from a few seconds to minutes and can be caused by epilepsy, fever, head injury, or other neurological conditions. Seizures vary in type and severity. Step-by-Step Explanation: How GABA Inhibition Leads to Epilepsy To understand how epilepsy is caused by inhibiting GABA, we can break the process into simple steps. These steps explain what GABA normally does, what changes happen when it is blocked and how those changes result in...
Read more

What is the cortical cytoskeleton?

The cortical cytoskeleton is a specialized and dynamic part of the cell's cytoskeleton located just beneath the plasma membrane. It is primarily composed of  actin filaments  (microfilaments) along with associated proteins like spectrin, ankyrin, filamin and ERM (ezrin, radixin, moesin) proteins. This region forms a dense network of filamentous proteins that help maintain the cell's shape, provide mechanical support and regulate interactions with the external environment. The cortical cytoskeleton plays a key role in cellular processes like endocytosis, cell motility, signal transduction and adhesion, especially in animal cells. Structure of the Cortical Cytoskeleton The main structural element of the cortical cytoskeleton is  F-actin,  which forms a thin, mesh-like layer closely attached to the inner surface of the plasma membrane. This layer is cross-linked by actin-binding proteins such as filamin and is anchored to membrane proteins via adaptor proteins like spec...
Read more

Write the name of two glands are the main secretary gland of brain

Image
The brain is not only the central organ of the nervous system but also contains two major secretory glands that are directly involved in endocrine functions. These are: Pituitary gland (Hypophysis) Pineal gland (Epiphysis cerebri) These glands are part of the neuroendocrine system, which links the brain and hormonal regulation. They help in maintaining homeostasis, regulating growth, metabolism, stress response, sleep cycle, reproduction and many other vital processes. 1. Pituitary Gland: The Master Gland The pituitary gland is called the  "master gland"  because it controls the activity of almost all other endocrine glands in the body. It is located at the base of the brain, in a bony cavity called the sella turcica of the sphenoid bone. It is connected to the  hypothalamus  by a stalk called the  infundibulum,  which carries signals from the brain to control hormone secretion. The pituitary gland has two main lobes: i. Anterior Pituitary (Adenohypophysis)...
Read more

Which neurological disorders are linked to increase dopamine secretion?

Dopamine is a crucial neurotransmitter in the brain that plays a significant role in controlling motor functions, emotional responses and reward pathways. It is involved in regulating mood, movement and several other physiological processes. However, when there is an imbalance in dopamine secretion or its activity, it can lead to several neurological and neuropsychiatric disorders. An increase in dopamine activity is linked to certain disorders, where excessive dopamine levels in specific brain regions can lead to abnormal behaviors and symptoms. Here are two neurological disorders closely associated with increased dopamine secretion: 1. Tourette Syndrome Tourette Syndrome (TS) is a neurological disorder characterized by repetitive, involuntary movements (motor tics) and sounds (vocal tics). It often begins in childhood and can persist into adulthood. In individuals with Tourette Syndrome, there is evidence of increased dopamine activity, particularly in the  basal ganglia,  a...
Read more

Which hormone is called as sleep hormone?

Image
The hormone that is called the sleep hormone is  Melatonin.  It is a naturally occurring hormone in the human body that controls the sleep-wake cycle, also known as the  circadian rhythm.  Melatonin is secreted by the  pineal gland,  which is a small, cone-shaped endocrine gland located deep in the brain, near the centre, just above the cerebellum and behind the third ventricle. Melatonin secretion is directly controlled by the amount of light the eyes receive. When the environment becomes dark, the retina sends signals to a special region in the hypothalamus known as the  suprachiasmatic nucleus (SCN).  This SCN then sends nerve signals to the pineal gland, which begins to release melatonin into the bloodstream. The rise in melatonin levels at night makes a person feel sleepy and relaxed, preparing the body for sleep. During the daytime, especially in the presence of sunlight or artificial light, melatonin secretion is stopped. This is why melato...
Read more

Write the name of neurotransmitter which act as neuromodulator as well as inhibitor of neurotransmitter

One of the best-known neurotransmitters that shows both neuromodulatory and inhibitory functions is  GABA (Gamma-Aminobutyric Acid).  It is a major chemical messenger in the central nervous system (CNS) and plays a vital role in regulating brain activity. GABA is unique because it not only participates in fast synaptic transmission as an inhibitory neurotransmitter but also acts more broadly as a neuromodulator that controls the excitability of entire neural circuits. 1. GABA as a Neuromodulator As a neuromodulator, GABA works at a slower and more widespread level than fast synaptic transmission. Instead of targeting a single postsynaptic neuron, it can influence large groups of neurons or entire regions of the brain. Neuromodulatory action of GABA usually happens through GABA-B receptors, which are metabotropic and linked with second messenger systems. Through these pathways, GABA can regulate the activity of other neurotransmitter systems like: Glutamate (main excitatory neu...
Read more