Transcript Document

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CHAPTER 3
CELL ENGINEERING
TECHNOLOGIES
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1. Cell fusion, hybridization, and mAb
2. PCR, RT-PCR and real time PCR
3. Gene cloning, expression, and gene expression detection
4. Gene mutation and genetic mutation
5. Gene knockout, RNAi, and transgenic animals
6. Clone of cell, embryo, and individual body
7. Gene map and human genome program
8. Stem cell technology, iPS
9. Immunological technologies
1. Cell fusion, hybridization, and mAb
1. Cell fusion: Two or more cells are combined (fused) to form one cell and
developed as one new cell clone or cell line.
2. Homokaryon fusion: The cell fusion between the cells with same gene types.
3. Heterokaryon fusion or hybridization fusion: The cell fusion between
the cells with different gene types.
4. Tumor hybridization: The cell fusion between cancer cells and normal cells
from different tissue types to form one hybridized cell with some special biological
characters or functions, such as cultured in vitro unlimitedly.
5. Hybridization antibody (mAb): Fuse cancer cells with some specific B
lymphocytes to form a hybridized cell clone to manufacture monoclonal antibody.
Milstein and Kohler won the 1984 Nobel Prize because they created mAb
technology in 1975.
Methods:
1.
2.
3.
4.
Operations under a microscope performance system
Chemical reagent (PEG)
Electron fusion
By viruses
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Unlimitedly cultured
and passaged
Mouse
Myeloma
Cell
+
Secret specific Ab
Mouse
B
Lymphocyte
=
A cell line can be cultured and
passaged unlimitedly in vitro
with specific Ab secretion
Hybridized Cell
Hybridization fusion by SV (sendai-virus)
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The advantages of mAb:
1. Very homogeneous and very specific even to a peptide because all of Ab
molecules are just from one B cell clone;
2. Easy to be prepared and obtain a big quantity.
The disadvantages of mAb:
1. Introduce the secondary Ab against itself because it is a powerful antigen to
the mAb receiver’s immune system;
2. The secondary Ab can result in severe super allergen to the mAb receiver.
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2. PCR and RT-PCR
PCR (Polymerase Chain Reaction) is the most creative
achievement in the molecular biology in past 30 years. “No
PCR, no life science today”. The PCR creator, Kary Mullis,
won the 1993 Nobel Prize on his great contribution.
PCR is a reaction that replicates the DNA fragment
following a DNA template in vitro, and can be designed and
controlled willfully to meet the investigator’s needs.
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Denaturation
Template
(94-95°C)
Primer
Annealing
(50-60°C)
(70-75°C)
New
synthesized
Strand
Recycles
PCR products
Extension
(25-35)
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PCR reaction system (Mixture):
DNA polymerase
Primer
Ion (Mg+)
dNTP
Template
Buffer
A PCR program:
1. T=95˚, 3′
3. T=72˚, 5′
5. T=52˚, 40"
7. GOTO 4 REP 36
9. HOLD 4˚
2. T=52˚, 40"
4. T=94˚, 1′
6. T=72˚, 2′
8. T=72˚, 10′
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Designation of PCR primers
What should be known well at least before design primers:
1. Template gene and its sequence
2. How long sequence you want to amplify from the template
3. Vector’s MCS and your restriction enzymes selection, which enzyme you can
choose, and which you can not
Basic steps for the primer designation:
1. Check template and select the length of the PCR product (If wanted by
project)
2. Select primer sequences and lengths, assemble them with restriction enzyme
sites and protection sequence together
3. Type the sequences into software, and check the Tm and GC ratio
4. Make your primer pair matched
5. Select best primer pair
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5’
3’
3’
5’
Upper XXXX
Lower XXXX
Sequence of EGFP, a template example:
CGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACG
GCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTG
ACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAC
CTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA
TGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCC
GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA
CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACA
AGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTC
GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCT
GAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG
TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAA
GCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGATCTAGATAA
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Upper I4 Nhe I:
5’-AAAAAAGCTAGCCGCCACCATGGTGAGCA-3’
(Length: 29bp, TM: 70.4˚C, GC: 51.7%)
Lower I4 Avr II:
5’-GGCCGGCCTAGGTTATCTAGATCCGGTGGA-3’
(Length: 30bp, TM: 70.4˚C, GC: 60%)
This primer designation is not so good because of:
1.Tm is too high. Usually, it should be at from 55°C to 65°C
2.The last nucleotide at 3’ is A that is easy to form wrong match
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A vector gene map and its MCS
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PCR machine (Thermocycler)
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3. Gene cloning, expression, and gene
expression detection:
Basic steps for a gene cloning project:
a. Amplify the target gene and clone it into a suitable vector
b. Transfect the recombinant into bacteria or eukaryotic cells to be
expressed
c. Screen the transfected bacteria or cells for positive clones
d. Detect the expression level, gene function, or harvest the expression
product
……
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Methods used to detect the gene expression:
Real time PCR: mRNA
Western blotting: Protein
Histochemistry: Protein distribution or location
Chemistry analysis: Changes of protein level and other linked features
Changes of the Morphology and function of the
expression host: Gene function
Others: Changes of the bio-functions of host cells or animal; Changes of
some special symptoms (for gene therapy), and other any changes based on
the target gene expression.
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4. Gene mutation and genetic mutation
Site directed mutation and multiple sites directed mutation.
Virtually, the mutation methods above are special PCR that
can result in one or more sites mutated.
Procedures:
1. Clone your target sequence into a plasmid and miniprep it from a dam+
E. coli strain, for example, DH5α
2. Primer designation
3. Run a mutation PCR with a powerful DNA polymerase
4. Use Dpn I to digest nonmutated dsDNA templates
5. Transfect bacteria with the reaction mixture and obtain clones
6. Miniprep of clones
7. Screen the correctly mutated clones by sequencing
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Primers designation for site directed mutation:
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Both of the mutagenic primers must contain the desired mutation and
anneal to the same sequence on opposite strands of the plasmid.
•
Primer length: 25 – 45mer
•
Mutated site should be in the middle of the primer with 10 – 15bp of correct
sequence on both sides.
•
Terminate in one or more C or G bases.
•
Purify the primer with FPLC.
•
Keep primer concentration in excess.
Primer example [Mutate “Ser” (AGC) into “Arg” (CGC)]:
Original template: 5´ CCA TGA TTA CGC CAA GAG CGC AAT TAA CCC TCA C 3’
Primers:
5´ CCA TGA TTA CGC CAA GCG CGC AAT TAA CCC TCA C 3’
5´ GTG AGG GTT AAT TGC GCG CTT GGC GTA ATC ATG G 3´
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Steps of the site
directed
mutation
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5. Gene knockout, RNAi, and transgenic animals (RNAi makes
gene knockdown, not knockout)
Gene knockout: (1) Construct a recombinant to insert some exogenous genes into an
exon of the genome to damage your target gene; (2) Transfect the recombinant into
cells or introduce it into embryonic cell and screen out the positive cell clones or
individuals to develop a gene knockout cell line or animal model.
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RNAi:
siRNA (19-25bp usually) can be understood as the follows: Small inhibiting
RNA, small interfering RNA, short inhibiting RNA, or short interfering RNA.
Synthesized siRNA
fragments
Transfect or introduce into
cells, tissue or animal
RNAi
Vector expressed
siRNA fragments
Detect the expression level of
the target gene, and observe
the phenotype of target gene
Both scientists from MIT and Stanford University won the Nobel prize in 2006
because of their great contribution for RNAi development.
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Transgenic animals:
The animal model with its some gene mutated, knockout, or some pathogenic
gene expressed.
6. Clone of cell, embryo, and individual body
Clone of cell:
Usually, limit dilution method is used to clonize cells.
Clone of embryo and individual body:
The genes of embryo cells were changed or mutated
New filial generations copulated each other (repeated)
Screen out the individuals with some genetic features designated
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7. Gene map and human genome program
Gene map means the sizes (lengths) and locations of all
genes and their control or regulation systems in a genome.
About human genome program.
About the Celera Genomics Group, a U.S. company I
visited many times.
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8. Immunological technologies
(1) Check or display Ag or Ab in cells and tissues
(2) Screen some library, such as, bio-panning
(3) Detect gene’s function and expression
(4) Classification of cell subtypes
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