Cellular Biology

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Cellular Biology
School of Life Sciences Shaanxi Normal University
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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

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• 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. Operations under a microscope performance
  system
• 2. Chemical reagent (PEG)
• 3. Electron fusion
• 4. By viruses

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A cell line can be cultured and passaged
unlimitedly in vitro with specific Ab secretion
Unlimitedly cultured and passaged
Secret specific Ab


Mouse Myeloma Cell
Mouse B Lymphocyte
Hybridized Cell
Hybridization fusion by SV (sendai-virus)
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• The advantages of mAb
• Very homogeneous and very specific even to a
  peptide because all of Ab molecules are just from
  one B cell clone
• Easy to be prepared and obtain a big quantity.
• The disadvantages of mAb
• Introduce the secondary Ab against itself because
  it is a powerful antigen to the mAb receivers
  immune system
• 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 investigators
needs.
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Template
Denaturation (94-95C)
Primer
Annealing (50-60C)
New synthesized Strand
Extension (70-75C)
Recycles (25-35)
PCR products
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PCR reaction system (Mixture) DNA polymerase
dNTP Primer

Template Ion (Mg)
Buffer A PCR
program 1. T95, 3'
2. T52, 40" 3. T72, 5'
4. T94, 1' 5.
T52, 40"
6. T72, 2' 7. GOTO 4 REP 36
8. T72, 10' 9. HOLD 4
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• Designation of PCR primers
• What should be known well at least before design
  primers
• Template gene and its sequence
• How long sequence you want to amplify from the
  template
• Vectors MCS and your restriction enzymes
  selection, which enzyme you can choose, and which
  you can not
• Basic steps for the primer designation
• Check template and select the length of the PCR
  product (If wanted by project)
• Select primer sequences and lengths, assemble
  them with restriction enzyme sites and protection
  sequence together
• Type the sequences into software, and check the
  Tm and GC ratio
• Make your primer pair matched
• Select best primer pair

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5
3
5
3
Upper XXXX
Lower XXXX
Sequence of EGFP, a template example CGCCACCATGGT
GAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGC
TGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAG
GGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG
CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCG
TGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTC
AAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA
GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACA
CCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGC
AACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTA
TATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC
GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAG
AACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCT
GAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACA
TGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGAC
GAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGC
AGTCGACGGTACCGCGGGCCCGGGATCCACCGGATCTAGATAA
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• Upper I4 Nhe I
• 5-AAAAAAGCTAGCCGCCACCATGGTGAGCA-3
• (Length 29bp, TM 70.4C, GC 51.7)
• Lower I4 Avr II
• 5-GGCCGGCCTAGGTTATCTAGATCCGGTGGA-3
• (Length 30bp, TM 70.4C, GC 60)
• This primer designation is not so good because
  of
• Tm is too high. Usually, it should be at from
  55C to 65C
• 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
• Clone your target sequence into a plasmid and
  miniprep it from a dam E. coli strain, for
  example, DH5a
• Primer designation
• Run a mutation PCR with a powerful DNA polymerase
• Use Dpn I to digest nonmutated dsDNA templates
• Transfect bacteria with the reaction mixture and
  obtain clones
• Miniprep of clones
• Screen the correctly mutated clones by sequencing

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• Primers designation for site directed mutation
• 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 genes function and expression (4)
Classification of cell subtypes