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cell engineering
This is a mind map about cell engineering, including research scope, applications, cell culture, cell fusion technology, etc.
Edited at 2023-11-24 01:35:58
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This is a mind map about plant asexual reproduction, and its main contents include: concept, spore reproduction, vegetative reproduction, tissue culture, and buds. The summary is comprehensive and meticulous, suitable as review materials.
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cell engineering
- bacteria
This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
- Plant asexual reproduction
This is a mind map about plant asexual reproduction, and its main contents include: concept, spore reproduction, vegetative reproduction, tissue culture, and buds. The summary is comprehensive and meticulous, suitable as review materials.
- Reproductive development of animals
This is a mind map about the reproductive development of animals, and its main contents include: insects, frogs, birds, sexual reproduction, and asexual reproduction. The summary is comprehensive and meticulous, suitable as review materials.
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- Outline
cell engineering
concept
It is a comprehensive science and technology that uses cell biology & molecular biology technology and engineering methods to study and transform biological genetic characteristics & biological characteristics at the cellular level, thereby obtaining specific cells, cell products or new biological varieties.
Research scope
Animal cell and tissue culture
Plant cell and tissue culture
Cell fusion (somatic cell hybridization)
Monoclonal antibodies
Nuclear transfer (cell disassembly)
chromosome engineering
animal
plant
embryo engineering
Stem cells and tissue engineering
cell bioreactor
application
Plant cell engineering
Seedlings detoxified & rapid propagation
Detoxification: getting rid of viruses, not toxins
basic research
somatic hybridization
Somatic cell fusion produces distant hybrids
Creation of special plant ploidy
Polyploidy
triploid culture
Endosperm culture
haploid
Anther culture
Pollen culture
Secondary product production
Cultivate new plant varieties
animal cell engineering
vaccine production
Hepatitis B surface antigen gene → inserted into mammalian cells → highly expressed → hepatitis B vaccine
Produce monoclonal antibodies
Detect very subtle strain-to-strain differences in multiple viruses → identify bacterial species & subspecies
Breeding fine varieties
Artificial insemination, embryo transfer and other technologies → animal husbandry production
interferon production
cell culture
concept
Animal, plant & microbial cells → sterile in vitro → grow, divide & reproduce, and no longer form tissues during culture.
plant cell culture
develop
cell theory
Schleiden & Schwann
The cortex of injured plant healing tissue can produce buds
cell totipotency theory
General steps
Material collection & sterilization
Disinfection enzymes: calcium hypochlorite, sodium hypochlorite, mercury chloride...
Prepare culture medium → Sterilization/sterilization → Aseptic operation → Inoculate biological materials into the culture medium
Put the culture medium into the incubator → Provide optimal culture conditions for the growth of various types of cells → When the cells reach a certain amount, harvest/passage them in time
medium
Usually includes: inorganic salts, carbon sources, vitamins, growth regulators, organic additives...
Carbon sources and energy
Sucrose & Glucose.
vitamins
Thiamine (required by plant cells in culture)
Niacin, pantothenic acid, biotin, folic acid
amino acids
L-Glutamine, proteolysis product.
plant growth hormone
Auxin
Indole Acetic Acid & Naphthalene Acetic Acid
mitogen
6-Benzylaminoadenine & Zeatin
Explant
Plant tissue culture: Plant material grown in vitro.
callus
Explant → tissue proliferation cells → a group of amorphous and sparsely arranged parenchyma cells, which are differentiated & have not formed a tissue structure.
induce
Choose the appropriate explant
Young embryo, hypocotyl, cotyledon
Choose the appropriate culture medium (MS, B5, N6)
Higher hormone concentrations
Rich organic add-ons
Higher content inorganic nitrogen source
Lower sucrose concentration
Appropriately shorten the interval between subcultures
Conducive to the formation of loose and friable callus.
suspension culture
Requirements for Suitable Suspension Culture
It has good looseness, fast proliferation and strong regeneration ability.
The appearance is generally bright milky white/light yellow in color, in the form of fine particles, loose and brittle.
VS suitable for regenerated plants
green
Screening and establishment of high-expression cell lines
Suspension culture has good dispersion, loose appearance and bright color.
Good uniformity
Fast growth and strong ability to synthesize secondary metabolites
single cell culture technology
Single cell isolation methods
mechanical method
Grind → Filtration & Centrifugation → Collection & Purification
Advantages: not damaged by enzymes, conducive to cell physiology & biochemistry research.
Disadvantages: Manual operation is difficult, and the number of complete cells obtained is small.
enzymatic method
Pectinase, cellulase
Osmoprotectant: Mannitol → to prevent enzyme damage to cells
Dextran sulfate potassium salt→increase the production of free cells
chemical method
Calcium oxalate → carrot suspension cell culture
Colchicine, 2,4-D (2,4-dichlorophenoxyacetic acid)/LH (luteinizing hormone)
Single cell culture methods
Nursing training
A piece of actively growing callus → promotes continued division & proliferation of cultured cells
Advantages and Disadvantages
Advantages: simple, easy and effective
Disadvantages: Cell division and growth processes cannot be tracked under a microscope.
plate culture
Suspend single cells & melted agar medium → mix → spread a thin layer on the bottom of the culture dish
Advantages and Disadvantages
advantage:
Uniform distribution → facilitates microscope: fixed-point observation of cells
High screening efficiency, large screening capacity and easy operation
Disadvantages: poor ventilation, easy accumulation of excreted substances → poisoning/affecting tissue absorption.
microchamber culture
Artificially manufactured sterile chamber: a drop of suspended cell solution → culture on a small amount of culture medium → division and proliferation → cell mass
Advantages and Disadvantages
Advantages: Continuous microscopic observation of cultured cells → Understand the growth, division, differentiation and other processes of a cell.
Disadvantages: less culture medium, difficulty in maintaining nutrients and water, large pH changes, and cultured cells can only divide for a short period of time.
Preservation of plant cells
Subculture and preservation methods
Low temperature storage method (5-10℃)
Ultra-low temperature storage method
Plant cells large scale culture
Applications in the food industry
Using plant cell engineering to produce spices
Production of food additives
Produce natural food
Features
Plant cells are sensitive to shear forces.
Plant cell cultures often form cell pellets.
Plant cells grow slowly and the operation cycle is long
Lots of foam.
Plant cells are pseudoplastic fluids when cultured at high concentrations.
The oxygen demand of plant cells is much lower than that of microorganisms.
Most plant cell cultures require light.
suspension culture
Suspend isolated plant cells in liquid culture medium for proliferation and culture.
Bioreactor
Mechanically stirred reactor
advantage
Biggest advantage: high dissolved oxygen content
Easier to control: temperature, pH, dissolved oxygen, nutrients
shortcoming
shear force problem
The power consumed per unit volume is larger than that of gas stirring
Stirring shaft→sterile seal
Non-mechanically stirred reactor Commonly used: gas mixer
air lift reactor
External circulation type
Internal circulation type
bubble reactor
Advantages and Disadvantages
advantage
Small shear force
Easy to keep sterile
Low operating costs
shortcoming
When cultivating in a highly sealed environment →mixing is not uniform enough
Excessive ventilation → easy elimination of CO2 & ethylene in the culture medium
Too high dissolved oxygen → not conducive to the synthesis of secondary metabolites
Advantage
1) The contact surface between cultured cells and culture substrate is large and the mass transfer effect is good.
2) It can take away harmful metabolites and avoid the problem of excessive local concentration of harmful products.
3) Can fully ensure the supply of oxygen.
immobilized culture
Plant cells → package: some polysaccharides/polymer compounds → culture & produce useful metabolites.
Bioreactor
packed bed reactor
fluidized bed reactor
membrane reactor
Advantage
(1) Conducive to the synthesis and accumulation of secondary substances;
(2) Reduce shear damage;
(3) Conducive to continuous culture & product collection.
animal cell culture
concept
Animal body→remove relevant tissues→disperse→single cells→appropriate culture medium→grow and proliferate & maintain function and structure.
Primary culture
Initial culture after digestion of animal tissue.
Passage culture
Trypsin → contact-inhibited adherent cells → detach from the culture bottle wall → cell suspension → aliquot → multiple culture bottles → continue the culture process.
cell lines
After the first cultured cells are successfully passaged.
cell lines
From a biologically identified cell line → use single cell isolation and culture or screening methods to form a cell population from single cells.
limited cell lines
The cell line has a limited survival period in vitro, that is, it cannot be passaged for a long time.
Infinite cell lines
It is a cell line that can survive continuously in vitro and has the ability to reproduce indefinitely.
Growth characteristics
adherent growth
adherent cells
Cells can grow only when they are attached to the surface of a solid support.
Most organisms' cells
suspension cells
Cells do not need to be attached to the surface of a solid support and can be grown in a suspended state.
tumor cells, white blood cells
contact inhibition
Cells: adherent growth → contact between cells → cell division & growth stop.
Tumor cells: none
density suppression
The cell density increases, the nutrients in the culture medium decrease, and the metabolites increase → nutrient depletion & impact of metabolites → cell division & growth stops
form
type
epithelial cell type
form
Similar to epithelial cells in the body, they are flat, irregular polygonal, and have round nuclei.
Growth characteristics
They are closely connected to each other and form a pavement-like thin layer; they move like a membrane when growing; they rarely break away from the cell group and move individually.
fibroblast type
form
The cell body is spindle-shaped or irregularly triangular; the cytoplasm extends outwards with 2-3 cell processes of varying lengths; there is an oval nucleus in the middle.
Growth characteristics
Arranged in a radial, swirling shape, not connected into sheets.
migrating cell type
form
The appearance is irregular and constantly changing, and dark phagocytic particles tend to appear within the cells.
Growth characteristics
The growth position is not fixed, scattered, and exhibits active wandering & deformation movements.
polymorphic cytotype
form
The shape is irregular, and the cells are composed of a slightly polygonal cell body and slender, pseudopod-like cell processes. Dark phagocytic granules are prone to appear inside the cells.
Factors influencing change
serum
pH
Cell density
Changes in growth status
Conversion or not
Growth & proliferation process of cultured cells
life span
The time that cells continue to proliferate & grow in culture medium.
include
Primary culture
Passage culture
Recession
Generation survival-growth curve
"Generation": the period of time from cell inoculation to isolation and culture.
include
incubation period
exponential growth period
equilibrium period
Recession
basic techniques
Sterile, non-toxic environment
Add a certain amount of antibiotics and replace the culture medium regularly.
Temperature and pH
The temperature is similar to the body temperature 35-37℃
pH 7.2~7.4.
Gas environment
O2 (required for cell metabolism)
CO2 (maintain pH)
CO2 incubator (95%O2 5%CO2)
nutrient content
Sugar, amino acids, growth-promoting factors, inorganic salts, trace elements, hormones, and animal or human serum...
Balanced salt solution BSS
Basic fluid for synthetic culture media
Used to wash cells.
animal cell large scale culture
Way
batch type
Streaming
Semi-continuous
Continuous
technology
suspension culture
adherent culture
immobilized culture
microcarrier culture technology
Large vector culture technology
porous carrier culture
Microencapsulation culture technology
Hollow fiber cell culture technology
cell fusion technology
concept
External force → two or more heterologous (species, genus) cells or protoplasts → contact with each other → without sexual process → membrane fusion, cytoplasmic fusion & nuclear fusion → phenomenon of forming hybrid cells.
process
Cells→fusion-promoting factors→agglutination phenomenon→plasma membrane adhesion→cytoplasmic fusion→nuclear fusion→hybrid cells.
induction method
Virus-induced cell fusion
Sendai virus, Newcastle disease virus, sporozoal virus...
The earliest fusion agent used: virus
Commonly used: inducing animal cell fusion
It needs to be inactivated with ultraviolet light or β-propiolactone first.
Mechanism: viral adhesion
Advantages and Disadvantages
advantage
High fusion rate, suitable for various animal cells
Easy to cultivate
shortcoming
Sendai virus unstable
The preparation process is complicated
After the virus is introduced into the cell, it may interfere with the life activities of the cell.
chemical fusion agent method
Polyethylene glycol (PEG) induction method
Mechanism: Change the membrane structure of various types of cells → evacuation & reorganization of lipid molecules in the membrane lipid bilayer at the contact point between two cells
Advantages and Disadvantages
advantage
Easy to use
Fusion frequency is higher
shortcoming
Have some toxicity
Egg cells Not applicable
NaNO3 treatment induces fusion
Mechanism: Na causes membrane potential change
Disadvantages: low fusion rate; harmful to highly vacuolated protoplasts from the mesophyll.
High pH-High Concentration Calcium Ion Treatment
Tobacco mesophyll protoplasts
Electrically induced cell fusion
Principle: Polarization into a dipole
step
① Electrophoresis
② Fusion
Advantages and Disadvantages
advantage
High fusion rate
Targeted induction of cell fusion under a microscope
Hybrid cells can be selected directly
Strong reproducibility and little damage to protoplasts
Exquisite device, convenient and simple
Eliminates the washing process after PEG induction
The induction process is highly controllable
shortcoming
Special cell electrofusion equipment must be purchased
Laser-induced cell fusion
The principle of optical tweezers
uniform electric field
non-uniform electric field
Optical tweezers pulling, assisting
Ion beam induced cell fusion
Cell surface is etched → permeability & transmembrane electric field change
protoplast
concept
Special method → removal of cell wall → exposed, viable protoplasm mass.
preparation
Material collection and disinfection
Draw materials
It can be used in various tissues & organs, but it is mostly used in leaves, callus & suspension cells; as well as shoot tips, cotyledons & embryonic tissues.
disinfect
Soapy water - 70% alcohol - 3% sodium hypochlorite - sterile water.
Isolation of protoplasts
mechanical separation method
Hypertonic sugar solution: pretreatment → slight plasmolysis → shrinkage into a spherical shape → mechanical grinding of tissue → wound release: intact protoplasts
Advantages and Disadvantages
Advantages: Avoids damage to protoplasts caused by enzyme preparations
Disadvantages: The number of complete protoplasts is small
Enzymatic separation method
Isotonic enzyme solution: degrade cell wall → keep warm for a certain period of time
Advantages and Disadvantages
Advantages: Large quantity available, widely applicable
Disadvantages: affects the vitality of protoplasts
Purification of protoplasts
flotation method
Advantages: It can avoid the separation of protoplasts from being damaged due to shock and impact by tissue fragments; the drugs used are simple and low cost.
Disadvantages: The requirements for centrifugal force are relatively strict; if the centrifugal force is not properly controlled, the protoplasts will not float easily.
Sedimentation method (filtration-centrifugation method)
Advantages: simple
Disadvantages: Causes breakage
discontinuous gradient method
Two solutions with different densities → form a discontinuous gradient → centrifuge → protoplasts & damaged cells → different liquid phases.
Advantages: A large number of pure protoplasts can be obtained; at the same time, it avoids the fragmentation of the protoplast plastids due to mutual extrusion during the collection process.
washing
protoplast culture medium
identification
hypotonic solution
Vitality assay
Pattern recognition
Fluorescence microscope identification method (FDA method)
FAD is non-fluorescent and non-polar, and can freely pass through the cytoplasmic membrane; it is hydrolyzed by lactonase in living cells → fluorescein (cannot freely pass through the plasma membrane).
√ Living cells: fluoresce
Phenol saffron staining method
√ Inactive protoplasts: red
Ivan blue staining
√ Dead cells & viable but damaged: stain
Identification of fusion elements
Microscopic identification
According to the difference in shape & structure of the protoplasts of the two parents →identify hybrids.
For example: basically colorless green → white-green: hybrid cells.
fluorescent label
Mechanical selection method based on visible marker traits.
FITC (fluorescein isothiocyanate) appears green under a fluorescence microscope.
RITC (Rhodamine Isothiocyanate) appears red under a fluorescence microscope
complementary selection method
Utilizing two parents with different genetic & physiological characteristics → specific culture conditions → only hybrid cells that complement each other can grow.
metabolic complementation
Utilize two auxotrophic/two resistant mutant protoplasts to fuse, and screen hybrid cells on a culture medium that is deficient in two substances/contains two harmful substances at the same time. The cell clusters that can grow can be initially considered to be hybrids. Cell formation.
growth complementation
The growth of parental protoplasts requires exogenous growth hormone, and hybrid cells formed by the fusion of parental protoplasts are autotrophic to growth hormone and can grow on media without exogenous growth hormone.
cell disassembly technology
cytoplasm
After removing the nucleus, it is an anucleated cell surrounded by a membrane. It is obtained by centrifugation after treatment with cytochalasin B.
nucleoplasm
The nucleus is separated from the cytoplasm, with a small amount of cytoplasm and a structure surrounded by a plasma membrane.
microcell
Cells with incomplete chromosomes in the nucleus.
cell reorganization
concept
Cell fusion technology & cell nucleoplasm separation technology → combination → fusion medium action → cytoplasm & complete cells / cytoplasm & nucleoplasm / micro cells & complete cells → process of re-constitution of cells.
three ways
Cell disassembly
concept
Intact cell nucleus & cytoplasm → special method (UV: kill the nucleus/absorb the nucleus) separation → homogeneous/differentiated nucleus & cytoplasm → recombine → cultivate new cells/organisms.
method
Physical disassembly method
Mechanical method or short wave light
Chemical disassembly method
Cytochalasin
nuclear transfer technology
Using micromanipulation technology to transplant the nucleus of one cell into another cell, or to exchange the nuclei (or cytoplasm) of two cells, it is possible to create asexual hybrids and new biological varieties.