ANSC 4050 Lecture Notes - Lecture 4: Hek 293 Cells, Lentivirus, Pre-Integration Complex
25 Sep 2018
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Lecture 4
→ Gene Therapy
Goal: to modify the genetic material of a patient’s cells for therapeutic purposes
• the genetic materials introduced are either expressed as proteins, interfering with other protein
expression, or aiming in gene editing
somatic cell gene therapy – to eliminate the clinical consequences of a disease and the inserted gene is
not passed on to the patient’s offspring
germ line gene therapy – gene modification or insertion in the fertilized egg of an animal; not ready in
human, ethical issues
→ Lentivirus
• a member of the retrovirus family
• mRNA genome virus
• most commonly used: HIV based
• pathogenic components deleted in vector
• transduce both dividing and non-dividing cell
• carries up to 8 kb foreign DNA
Major Structure of HIV:
• a diploid single stranded positive sense RNA-genome; approx. 10 kb long
• Gp120: binds to T cell CD4 receptor and CCR5 receptor then triggers cell entry
• pre-integration complex main components: integrase (the product of the vpr gene), matrix
(product of the gag gene)
→ HIV based gene therapy
transfer vector –
a) cis-acting genetic sequences for the vector to infect the target cell
b) for transfer of the therapeutic (or reporter) gene, contains restriction sites for insertion of desired
genes
packaging vector – allows production of proteins essential for transcription and packaging of RNA,
packing the expression construct into recombinant pseudoviral particles
-many HIV genes are deleted, thus the virus is unable to reproduce after it infected the new host
(targeting) cell
packaging cell – HEK293 cell, a human embryonic kidney cell line; a adenovirus transformed cell with
some virus protein expressing gene, an insertion of approx. 4.5 kb from the left arm of the viral genome
in chromosome 19
→ Safety considerations of lentiviral vector
• packaging plasmid does not contain the necessary HIV genes
• absence of replication competent lentiviruses
• not allowing for replication of HIV in humans
Precaution: HIV may be produced during manufacture of the vector in the packaging cell line if
unexpected recombination
→ Lentiviral Vector Applications
1. Genome-wide functional studies: combined with ShRNA, siRNAs – panels for high-throughput
loss of function screens
2. Animal transgenesis
3. Gene therapy
-about 54 gene therapy clinical trial using LVs are ongoing or have been approved
→ Types of plasmid vector
• Cloning (shuttle) vector
• Expression vector
Recombinant Protein Expression Vector Components –
P = promoter
MCS = multiple cloning sites
polyA = poly A signal
Neo – Neomycin resistant gene
→ Expression of Two Protein Simultaneously
bicistronic vector – a construct with two genes separated from each other by IRES sequence, allow
expression of two proteins simultaneously
IRES (internal ribosomal entry site) – found in mammalian virus genome, allow simultaneous translation
of different proteins from a bicistronic mRNA molecule
→ Fusion Protein
• protein created through the joining of two or more genes in frame which originally coded for
separate proteins
• translation of this fusion gene results in a single protein with properties derived from each of
the original proteins
Promoter
cDNA for POI
cDNA of fusion partner
POI: protein of interest
Document Summary
Lentivirus: a member of the retrovirus family, mrna genome virus, most commonly used: hiv based, pathogenic components deleted in vector carries up to 8 kb foreign dna transduce both dividing and non-dividing cell. Major structure of hiv: a diploid single stranded positive sense rna-genome; approx. 10 kb long: gp120: binds to t cell cd4 receptor and ccr5 receptor then triggers cell entry, pre-integration complex main components: integrase (the product of the vpr gene), matrix (product of the gag gene) 4. 5 kb from the left arm of the viral genome in chromosome 19. Safety considerations of lentiviral vector: packaging plasmid does not contain the necessary hiv genes, absence of replication competent lentiviruses, not allowing for replication of hiv in humans. Precaution: hiv may be produced during manufacture of the vector in the packaging cell line if unexpected recombination. Lentiviral vector applications: genome-wide functional studies: combined with shrna, sirnas panels for high-throughput loss of function screens, animal transgenesis, gene therapy.
