Skip to main content

Microcell Fusion Technique – Detailed Notes


Microcell Fusion Technique – Detailed Notes


1. Definition 

Microcell fusion is a technique in which microcells containing isolated chromosomes or chromosomal fragments from a donor cell are fused with recipient cells.
It is used for chromosome transfer, gene mapping, and creating hybrid cell lines.
Also called microcell-mediated chromosome transfer (MMCT).
2. Principle
Donor cells containing the chromosome of interest are treated with colchicine to induce micronuclei formation.
These micronuclei are isolated as microcells (small cytoplasmic vesicles containing one or a few chromosomes).
Microcells are fused with recipient cells using fusion agents such as PEG or Sendai virus.
The chromosome from the donor is incorporated into the recipient cell, creating a stable hybrid.
Key Concept:
Microcell fusion allows transfer of specific chromosomes from donor to recipient without introducing the whole genome.


3. Steps in Microcell Fusion Technique


Step 1: Preparation of Donor Cells

Donor cells contain the chromosome(s) of interest, often labeled with selectable markers.
Cells are treated with colchicine, which disrupts mitotic spindle formation.
This produces micronuclei, small nuclear fragments containing single chromosomes.

Step 2: Isolation of Microcells

Micronucleated donor cells are treated with cytochalasin B, which inhibits cytoplasmic division.
Cells are centrifuged through a Percoll density gradient to isolate microcells.
Microcells are small vesicles (~2–5 µm) containing single or few chromosomes.

Step 3: Fusion with Recipient Cells


Microcells are fused with recipient cells using:
A. PEG-Mediated Fusion
Polyethylene glycol induces membrane fusion between microcells and recipient cells.
B. Virus-Mediated Fusion
Inactivated Sendai virus is used to facilitate fusion of microcell membranes with recipient cells.

Step 4: Selection of Hybrid Cells

Recipient cells receiving the donor chromosome are selected using selectable markers (e.g., antibiotic resistance, reporter genes).
Hybrid cells are then expanded in culture.

Step 5: Analysis of Hybrid Cells

Hybrid cells are analyzed for chromosome retention, gene expression, and functional traits.
Techniques include:
Karyotyping
Fluorescent in situ hybridization (FISH)
PCR or molecular markers

4. Factors Affecting Microcell Fusion

Donor cell preparation – Colchicine concentration and treatment duration
Recipient cell type – Must be compatible and capable of growth
Fusion method – PEG concentration, viral activity, incubation time
Selection strategy – Choice of selectable markers
Microcell viability – Critical for successful fusion
5. Applications of Microcell Fusion Technique

5.1 Gene Mapping


Transfer individual chromosomes to recipient cells for mapping gene loci.
Example: Transfer human chromosome 21 into hamster cells for mapping Down syndrome-related genes.

5.2 Functional Genomics

Study gene function by isolating and expressing single donor chromosomes in a new cellular environment.

5.3 Production of Hybrid Cell Lines

Generate somatic cell hybrids with specific chromosomes for research.
Example: Human-mouse hybrid cell lines for gene expression studies.

5.4 Disease Research


Study genetic diseases by transferring mutant chromosomes into recipient cells.
Example: Identify genes responsible for metabolic disorders.

5.5 Chromosome Engineering

Combine specific donor chromosomes with recipient cells for gene transfer and therapeutic research.

6. Advantages of Microcell Fusion


Transfer specific chromosomes or chromosomal fragments
Bypasses sexual or species barriers
Produces stable hybrid cell lines
Useful in gene mapping and functional genomics
Can introduce large DNA fragments that are difficult with plasmids or viral vectors

7. Limitations
Technically demanding and labor-intensive
Low efficiency of microcell fusion
Requires careful donor cell preparation
Hybrid cells may lose donor chromosomes over time
Requires selectable markers for identification

8. Examples
Donor Chromosome
Recipient Cell
Purpose
Human chromosome 21
Chinese hamster ovary (CHO) cells
Gene mapping, Down syndrome studies
Human chromosome 7
Mouse cells
Functional genomics, disease gene identification
Specific donor chromosome
Fibroblast cells
Production of hybrid cell lines for research

Advantages
Transfer specific chromosomes, bypass sexual barriers, stable hybrids.

Limitations
Technically demanding, low efficiency, requires selection



Microcell Fusion Technique – 50 MCQs


Microcell fusion is used to:
a) Fuse plant protoplasts
b) Transfer donor chromosomes to recipient cells ✅
c) Fuse viral particles
d) Introduce RNA only
Microcell fusion is also called:
a) Somatic hybridization
b) Microcell-mediated chromosome transfer (MMCT) ✅
c) DNA-mediated transformation
d) Lipofection
Microcells are:
a) Whole donor cells
b) Cytoplasmic vesicles containing one or few chromosomes ✅
c) Viral particles
d) Plasmids
Donor cells are treated with which chemical to induce micronuclei?
a) PEG
b) Colchicine ✅
c) Cytochalasin B
d) Cellulase
Cytochalasin B is used to:
a) Induce fusion
b) Prevent cytoplasmic division and isolate microcells ✅
c) Digest cell walls
d) Label chromosomes
Microcells contain:
a) Entire genome
b) One or few chromosomes ✅
c) Only RNA
d) Only cytoplasm
Fusion of microcells with recipient cells can be done by:
a) PEG-mediated fusion ✅
b) Electroporation
c) Lipofection
d) Calcium phosphate precipitation
Virus-mediated fusion uses:
a) Active viruses
b) Inactivated Sendai virus ✅
c) Retrovirus
d) Adenovirus
Microcell fusion is primarily applied in:
a) Plant protoplast fusion
b) Chromosome transfer and hybrid cell production ✅
c) Bacterial transformation
d) RNA interference
Selectable markers are important in microcell fusion to:
a) Induce fusion
b) Identify successful hybrid cells ✅
c) Digest cell walls
d) Label chromosomes
Hybrid cells generated by microcell fusion contain:
a) Only recipient chromosomes
b) Donor chromosome(s) plus recipient genome ✅
c) Only donor chromosomes
d) Only cytoplasm
Donor cell chromosomes may be labeled with:
a) Fluorescent markers ✅
b) PEG
c) Colchicine
d) Lipid vesicles
Density gradient centrifugation is used to:
a) Fuse microcells
b) Isolate microcells ✅
c) Select hybrid cells
d) Digest cell walls
PEG (Polyethylene Glycol) in microcell fusion acts by:
a) Digesting cell walls
b) Destabilizing membranes to allow fusion ✅
c) Selecting hybrid cells
d) Labeling chromosomes
Which of the following is a key advantage of microcell fusion?
a) Transfers only RNA
b) Transfers specific donor chromosomes ✅
c) Works without donor cell preparation
d) Requires no selection
Microcell fusion bypasses:
a) Sexual reproductive barriers ✅
b) Cell culture
c) DNA transfer
d) Protein synthesis
Donor cells must be treated with colchicine for:
a) 1–2 minutes
b) Several hours to induce micronuclei formation ✅
c) Only to label chromosomes
d) To digest the cytoplasm
Microcells are typically:
a) 20–50 µm
b) 2–5 µm ✅
c) 10–15 µm
d) 50–100 µm
Cytochalasin B inhibits:
a) DNA replication
b) Cytokinesis ✅
c) Chromosome segregation
d) Nuclear division
Hybrid cell lines created by microcell fusion are useful for:
a) Gene mapping ✅
b) Functional genomics ✅
c) Disease research ✅
d) All of the above ✅
Microcell fusion efficiency depends on:
a) Donor cell preparation ✅
b) Recipient cell type ✅
c) Fusion method and incubation conditions ✅
d) All of the above ✅
Hybrid cells can be analyzed using:
a) Karyotyping ✅
b) FISH (Fluorescent in situ hybridization) ✅
c) PCR ✅
d) All of the above ✅
Microcell fusion can transfer:
a) Nuclear chromosomes ✅
b) Cytoplasmic organelles
c) Only DNA plasmids
d) Only RNA
Donor chromosomes may carry:
a) Selectable markers ✅
b) Antibiotic resistance genes ✅
c) Reporter genes ✅
d) All of the above ✅
Microcell fusion is technically:
a) Simple and fast
b) Labor-intensive and demanding ✅
c) Only for plants
d) Only for bacteria
Hybrid cells are selected based on:
a) Morphology
b) Marker gene expression ✅
c) Chromosome number
d) Cell size
Microcell fusion can be applied to study:
a) Gene function ✅
b) Disease mechanisms ✅
c) Chromosome mapping ✅
d) All of the above ✅
Donor cells for microcell fusion are often:
a) Tumor cells ✅
b) Plant cells
c) Bacterial cells
d) Viral particles
Which step is critical for microcell viability?
a) Colchicine treatment duration ✅
b) Fusion method
c) Selectable marker choice
d) PCR analysis
Hybrid cells may lose donor chromosomes over time due to:
a) Culture instability ✅
b) Fusion method
c) PEG concentration
d) Colchicine treatment
Microcell fusion can transfer:
a) Single chromosomes ✅
b) Multiple chromosomes ✅
c) Entire genome
d) Both a & b ✅
Microcell fusion is used in human genetics to:
a) Study Down syndrome genes ✅
b) Study plant traits
c) Bacterial cloning
d) Viral replication
Hybrid cells can express genes from:
a) Donor chromosomes ✅
b) Recipient chromosomes ✅
c) Both donor and recipient ✅
d) None
A limitation of microcell fusion is:
a) High efficiency
b) Requires careful donor cell preparation ✅
c) Works without selection
d) Can transfer RNA only
Sendai virus in microcell fusion functions as:
a) Selectable marker
b) Fusion agent ✅
c) Donor chromosome carrier
d) Cytoplasmic stabilizer
Hybrid cells are maintained in culture using:
a) Selective medium ✅
b) PEG only
c) Colchicine only
d) Viral infection
Applications of microcell fusion in cancer research include:
a) Mapping oncogenes ✅
b) Generating hybrid cell lines ✅
c) Functional genomics ✅
d) All of the above ✅
Microcell fusion can transfer which types of chromosomes?
a) Autosomes ✅
b) Sex chromosomes ✅
c) Fragmented chromosomes ✅
d) All of the above ✅
Hybrid cells can be used to study:
a) Drug resistance ✅
b) Gene expression ✅
c) Cytogenetic abnormalities ✅
d) All of the above ✅
Microcell fusion differs from protoplast fusion because:
a) It transfers whole cells
b) It transfers only chromosomes, not whole cells ✅
c) It uses plant tissues only
d) It uses viral vectors
Key chemical for micronuclei induction is:
a) PEG
b) Colchicine ✅
c) Cytochalasin B
d) Calcium chloride
PEG is used in microcell fusion to:
a) Digest cell walls
b) Promote membrane fusion ✅
c) Select hybrid cells
d) Label chromosomes
Donor chromosomes may be marked with:
a) Fluorescent probes ✅
b) Antibiotic resistance genes ✅
c) Reporter genes ✅
d) All of the above ✅
Density gradient centrifugation isolates:
a) Donor chromosomes
b) Microcells ✅
c) Recipient nuclei
d) Viral particles
Microcell fusion can produce stable hybrids for:
a) Long-term gene mapping ✅
b) Temporary expression
c) Only protoplast studies
d) Only viral vectors
Hybrid cells can retain donor chromosomes depending on:
a) Culture conditions ✅
b) Donor chromosome type ✅
c) Selective pressure ✅
d) All of the above ✅
Microcell fusion is used in mapping which human chromosome associated with Down syndrome?
a) Chromosome 21 ✅
b) Chromosome 7
c) Chromosome 1
d) Chromosome X
Hybrid cells from microcell fusion are analyzed by:
a) Karyotyping ✅
b) PCR ✅
c) FISH ✅
d) All of the above ✅
A major advantage of microcell fusion is:
a) Transfers whole donor genome
b) Transfers specific chromosomes ✅
c) No selection required
d) Works only in plants
Overall, microcell fusion is important in:
a) Gene mapping ✅
b) Functional genomics ✅
c) Disease research ✅
d) All of the above ✅


Comments

Post a Comment

Popular Posts

Protein Structure Database (PDB)

Protein Structure Database (PDB) Introduction The Protein Structure Database (PDB) is the primary global repository for the three-dimensional (3D) structures of biological macromolecules such as proteins, nucleic acids, and protein–ligand complexes. These structures are determined experimentally using techniques like X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and Cryo-Electron Microscopy (Cryo-EM). PDB plays a vital role in understanding: Protein structure and function Molecular interactions Drug discovery and design Structural biology and bioinformatics History and Development Established in 1971 Founded by Brookhaven National Laboratory (USA) Initially contained only 7 protein structures Now maintained by the Worldwide Protein Data Bank (wwPDB) Members of wwPDB RCSB PDB (USA) PDBe (Europe) PDBj (Japan) BMRB (Biological Magnetic Resonance Data Bank) Objectives of PDB To collect, store, and distribute 3D structural data of biomolecules To provide free and ope...
Post a Comment
Read more

❥ Southern Blotting Notes

Southern Blotting  ❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥  Introduction Southern blotting is a molecular biology technique used for the detection of specific DNA sequences in a complex mixture of DNA. It was developed by Edwin M. Southern in 1975. The method involves restriction digestion of DNA, separation by gel electrophoresis, transfer (blotting) onto a membrane, and hybridization with a labeled DNA probe. Principle of Southern Blotting The technique is based on the principle of complementary base pairing. A single-stranded labeled DNA probe hybridizes specifically with its complementary DNA sequence immobilized on a membrane. Detection of the label confirms the presence and size of the target DNA fragment. Steps Involved in Southern Blotting. 1. Isolation of DNA Genomic DNA is extracted from cells or tissues. DNA must be pure and intact to ensure accurate results. 2. Restriction Enzyme  Digestion DNA is digested using specific restriction endonucleases. Produces DNA f...
1 comment
Read more

𓆞 Western Blotting Notes

Western Blotting (Immunoblotting) ❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥  Introduction Western blotting, also known as immunoblotting, is a widely used analytical technique for the detection, identification, and quantification of specific proteins in a complex biological sample. The technique combines protein separation by gel electrophoresis with specific antigen–antibody interaction. The method was developed by Towbin et al. (1979) (Burnette 1981---its group work) and is called “Western” in analogy to Southern blotting (DNA) and Northern blotting (RNA). Principle The principle of Western blotting involves: Separation of proteins based on molecular weight using SDS-PAGE Transfer (blotting) of separated proteins onto a membrane Specific detection of the target protein using primary and secondary antibodies Visualization using enzymatic or fluorescent detection systems 👉 Antigen–antibody specificity is the core principle of Western blotting. Steps Involved in Western Blotting 1. Sa...
Post a Comment
Read more

✩‧₊ Plaque Blotting Technique

Plaque Blotting Technique *ੈ✩‧₊˚༺☆༻*ੈ✩‧₊˚*ੈ✩‧₊˚༺☆༻*ੈ✩‧₊˚ Introduction Plaque blotting is a molecular biology screening technique used to identify specific DNA or RNA sequences present in bacteriophage plaques formed on a bacterial lawn. It is especially useful in the screening of recombinant phage libraries such as λ (lambda) phage genomic or cDNA libraries. This technique combines: Plaque assay (to isolate individual phage clones) Blotting technique (to transfer nucleic acids onto a membrane) Hybridization (to detect specific sequences using labeled probes) Principle of Plaque Blotting The principle of plaque blotting is based on nucleic acid hybridization. Each plaque represents a clone of phage particles containing identical DNA. DNA from phage particles in plaques is: Released Denatured into single strands Transferred onto a nitrocellulose or nylon membrane The membrane is incubated with a labeled DNA/RNA probe complementary to the target sequence. Hybridization between probe and t...
Post a Comment
Read more

RESTRICTION MAPPING

RESTRICTION MAPPING Introduction Restriction mapping is a molecular biology technique used to determine the relative positions of restriction enzyme recognition sites on a DNA molecule. It involves digestion of DNA with one or more restriction endonucleases followed by analysis of fragment sizes using agarose gel electrophoresis. Restriction mapping is essential for DNA characterization, cloning strategies, gene localization, and genome analysis. Definition Restriction mapping is the process of identifying the number, order, and distances between restriction enzyme cleavage sites within a DNA fragment by analyzing the pattern of fragments generated after enzymatic digestion. Principle Restriction enzymes cut DNA at specific palindromic nucleotide sequences. When DNA is digested with: Single restriction enzyme → produces fragments based on its recognition sites Multiple restriction enzymes → produces fragments whose sizes reveal the relative positions of sites By comparing fragment size...
Post a Comment
Read more

DNA FOOTPRINTING

DNA FOOTPRINTING Introduction DNA footprinting is a molecular biology technique used to identify the specific site(s) on DNA where proteins (such as transcription factors) bind. It reveals the exact nucleotide sequences protected by bound proteins against cleavage by nucleases or chemical agents. It is widely used to study DNA-protein interactions, transcription regulation, and gene expression control. Definition DNA footprinting: A technique used to locate the binding site of DNA-binding proteins on DNA by detecting protected regions that are resistant to enzymatic or chemical cleavage. Principle DNA-binding proteins protect the DNA segment they occupy. DNA exposed to nucleases (DNase I) or chemical cleavage agents is cut at accessible regions. Regions bound by protein remain unaffected, leaving a “footprint”. When fragments are separated on a denaturing polyacrylamide gel, the missing bands correspond to protein-binding sites. Key idea: Cleavage occurs everywhere except where the pro...
Post a Comment
Read more

••CLASSIFICATION OF ALGAE - FRITSCH

      MODULE -1       PHYCOLOGY  CLASSIFICATION OF ALGAE - FRITSCH  ❖F.E. Fritsch (1935, 1945) in his book“The Structure and  Reproduction of the Algae”proposed a system of classification of  algae. He treated algae giving rank of division and divided it into 11  classes. His classification of algae is mainly based upon characters of  pigments, flagella and reserve food material.     Classification of Fritsch was based on the following criteria o Pigmentation. o Types of flagella  o Assimilatory products  o Thallus structure  o Method of reproduction          Fritsch divided algae into the following 11 classes  1. Chlorophyceae  2. Xanthophyceae  3. Chrysophyceae  4. Bacillariophyceae  5. Cryptophyceae  6. Dinophyceae  7. Chloromonadineae  8. Euglenineae    9. Phaeophyceae  10. Rhodophyceae  11. Myxophyce...
Post a Comment
Read more

Electroporation – Detailed Notes

Electroporation – Detailed Notes Definition : Electroporation is a physical method of gene transfer in which cells are exposed to a brief, high-voltage electric pulse, creating temporary pores in the cell membrane. This allows DNA, RNA, proteins, or other molecules to enter the cytoplasm. It is widely used in bacteria, yeast, plant protoplasts, and mammalian cells. Key Concept: The electric field destabilizes the membrane, making it permeable to macromolecules. 1. Principle Cells are suspended in a conductive medium. A brief electrical pulse induces transient pores in the plasma membrane. DNA or other molecules present in the medium enter the cell through these pores. Membrane reseals after the pulse, and the molecule is retained inside the cell. Advantages of Principle: Direct and rapid. Works in many cell types. Does not require chemical carriers or viral vectors. 2. Materials Required Cells – bacterial, yeast, plant protoplasts, mammalian cells. DNA/RNA/other macromolecule – purifie...
Post a Comment
Read more

Fourth Semester M.Sc. Degree Examination, September 2019BotanySpecial Paper II - ElectiveBO 242 a: BIOTECHNOLOGY(2013 Admission onwards)

Reg. No.......  Name......... G-5263 Fourth Semester M.Sc. Degree Examination, September 2019 Botany Special Paper II - Elective BO 242 a: BIOTECHNOLOGY (2013 Admission onwards) Max. Marks: 75 1. Answer the following questions: 1. Humulin 2. YAC 3. Cybrids 4. Hybridomas 5. IPR 6. Gene therapy 7. C DNA library 8. AFLP 9. Hairy root culture 10. Somacional variation (10 x 1=10 Marks) II. Answer the following questions in not more than 50 words : 11. (a) What are immobilized enzymes? What is its advantage? OR (b) Write a short note on molecular farming. 12. (a) Give an account of bioprocess technology for the production of secondary metabolites. OR (b) What are bioreactors? How it operates? 13. (a) What are probiotics?. How do they work? OR (b) Discuss the methodology and application of western blotting. 14. (a) Briefly explain the application of protoplast culture OR (b) Write a short note on gene therapy 15. (a) What are reporter genes? Discuss its utility in transformation studies O...
Post a Comment
Read more

Information retrieval from databases - search concepts, Tools for searching, homology searching, finding Domain and Functional site homologies

Information retrieval from databases - search concepts, Tools for searching, homology searching, finding Domain and Functional site homologies Information Retrieval from Databases 1. Introduction Information retrieval in bioinformatics refers to the process of extracting relevant biological data (DNA, RNA, protein sequences, structures, or functional information) from databases. Aim : Identify sequences, functions, or structural features for analysis, comparison, and annotation. Databases can be primary (raw sequence data) or secondary/derived (annotated, processed data). 2. Search Concepts in Biological Databases 2.1 Types of Searches Exact Match Search Returns results only if the query exactly matches database entries. Useful for known accession numbers or IDs. Pattern/Keyword Search Searches based on specific motifs, keywords, or annotations. Example: “kinase domain,” “signal peptide.” Similarity/Homology Search Detects sequences similar to the query based on sequence alignment. Use...
Post a Comment
Read more