Lecture 9: Recombinant DNA Technology and Genetic Engineering

Microbial  Gene-cs  
BI  302  
Instructor:  Dr.  Nalina  Nadarajah  
Email:  nnadarajah@centennialcollege.ca  
 Agenda  for  This  Week  

• An  introduc-on  to    
– Recombinant  DNA  technology  (online  module)    

– Gene4c  engineering  (in-­‐class)  

2  
 Overview  
• Recombinant  DNA:  DNA  created  by  joining  together  pieces  of  
DNA  from  different  sources    
• Recombinant  DNA  technology  exploits  features  of  genes,  gene  
expression,  and  DNA  enzymology  to  create  novel  DNA  
molecules  for  study  
• Although  natural  processes  such  as  crossing  over  produce  
recombined  DNA  molecules,  the  term  usually  refers  to  ar4ficial  
and  deliberate  recombina4on  of  pieces  of  DNA  from  different  
organisms  that  are  not  found  together  in  nature  
• Ac-ve  growth  since  the  early  1970s  
• Gene-c  engineering  applies  recombinant  DNA  technology  to  
problems  in  biology,  medicine,  and  agriculture  
3  
 Overview  
• Recombinant  DNA  technology  began  with  2  key  tools:  

• Recombinant  DNA  is  made  by  splicing  a  foreign  DNA  fragment  

into  a  cloning  vector  (small  replica-ng  molecule  such  as  a  
bacterial  plasmid),  which  will  then  amplify  that  fragment  
along  with  itself  and  result  in  a  molecular  clone  of  the  
inserted  DNA  

• Restric4on  enzymes  cut  DNA  at  specific  target  sites,  resul-ng  

in  defined  fragments  with  s-cky  ends  suitable  for  inser-on  
into  a  vector  that  has  been  cut  open  by  the  same  enzyme    

4  
 Overview  Cont.  
• Foreign  DNA  is  spliced  into  a  vector  for  amplifica-on,  
producing  a  clone  of  the  inserted  DNA  
• Restric-on  endonucleases  cut  DNA  at  specific  target  sites  
• Polymerase  chain  reac-on  (PCR)  can  be  used  for  specific  
DNA  amplifica-on  
• Labeled  single-­‐stranded  DNA  or  RNA  can  be  used  as  a  probe  
to  iden-fy  molecules  containing  its  base-­‐pair  complement  
• Virtually  any  nucleo-de  sequence,  including  restric-on  sites  
can  be  mapped  
• DNA  can  be  sequenced  
• Transgenes  can  be  constructed  and  expressed  in  foreign  
hosts  
5  
 From  Bacteriophages  to  Restric-on  
Enzymes  
• mid  1960’s  –  Werner  Arber  showed  E.coli  produces  
restric-on  endonucleases  (works  as  pair  of  molecular  
scissors)  and  modifica+on  enzyme  which  adds  methyl  groups  
to  DNA  hence  preven-ng  restric-on  endonuclease  ac-vity  
• 1970  –  Hamilton  Smith  isolated  HindIII  from  Haemophilus  
influenza  (blunt  end  cucer)  
• 1973  –  Daniel  Nathans  created  first  restric-on  map  
• 1975  –  Paul  Berg  created  first  recombinant  DNA  molecule  
• Arber,  Smith  and  Nathans  shared  the  Nobel  Prize  in  1978  
• Berg  won  the  Nobel  Prize  in  1980  
6  
 Restric-on  Enzymes  
• Produced  by  bacteria  as  a  defense  mechanism  against  
infec-ons  by  viruses  
- more  than  3500  restric-on  enzymes  have  been  iden-fied  

• Recognizes  and  binds  to  DNA  at  a  specific  nucleo-de  

sequence  known  as  a  restric-on  site  
- enzyme  (molecular  scissors)  cuts  both  strands  of  DNA  
within  that  sequence  by  cleaving  the  phosphodiester  
bonds  
- also  known  as  endonucleases  

• Most  recogni-on  sequences  display  symmetry  (palindrome)  

- usually  a  4-­‐base  pair  or  6-­‐base  pair  cucer   7  
 Common  Restric-on  Enzymes  

8  

Photo Source: Fig. 20-1 from Concepts of Genetics 10th Edition by Klug et al., 2012
 Crea-on  of  Recombinant  DNA  

9  

Photo Source: Fig. 20-2 from Concepts of Genetics 10th Edition by Klug et al., 2012
 Vectors  
• Vectors  carry  DNA  molecules  to  be  cloned  

• Vectors  are  carrier  DNA  molecules  that  can  replicate  cloned  

DNA  fragments  in  a  host  cell  

• Vectors  must  be  able  to  replicate  independently  and  should  

have  several  restric-on  enzyme  sites  to  allow  inser-on  of  a  
DNA  fragment  

10  
 Key  Proper-es  of  Vectors  
• Size    
– must  be  small  enough  to  separate  easily  
• Mul-ple  cloning  sites  (MCS)    
– a  stretch  of  DNA  with  recogni-on  sequences  for  common  
restric-on  enzymes  
• Origin  of  replica-on  (ori)    
– DNA  sequence  at  which  replica-on  is  ini-ated  
• Selectable  marker  genes    
– allow  for  selec-on  and  iden-fica-on  of  transformed  bacteria  
• RNA  polymerase  promoter  sequences    
– place  where  RNA  polymerase  binds  to  begin  transcrip-on  
• DNA  sequencing  primer  sequences  
– known  sequence  that  allows  sequencing  of  cloned  DNA   11  
 Examples  of  Vectors  
1. Bacterial  plasmids  
2. Bacteriophage  vectors  
3. Cosmid  vectors  
4. Bacterial  ar-ficial  chromosomes  (BACs)  
5. Yeast  ar-ficial  chromosomes  (YACs)  
6. Tumor-­‐inducing  (Ti)  vectors  

12  
 1.  Bacterial  Plasmid  Vectors  
• First  vectors  developed  and  s-ll  widely  used  
• Plasmid  DNA  is  circular,  double  stranded,  self-­‐replica-ng  
DNA  that  scien-sts  can  manipulate  to  carry  and  clone  other  
pieces  of  DNA  
– found  primarily  in  bacteria  

• Introduced  into  hosts  by  transforma-on  

• pBR  plasmids  (-­‐ve  selec-on)  or  pUC  plasmids  (+ve  selec-on)  
• Primary  limita-on  of  bacterial  plasmids  as  vectors  is  the  size  
of  DNA  fragments  (usually  cannot  exceed  6-­‐7kb)  

13  
Two  Plasmids  as  Vectors  for  DNA  Cloning  

ampR tetR

14  
Nega4ve  Selec4on  –  pBR322  

15  
Posi4ve  Selec4on  –  pUC  vector  

16  
 Cloning  with  Bacterial  Plasmid  

Photo Source: Fig. 20-5 from Concepts of Genetics 10th Edition by Klug et al., 2012
Posi4ve  Selec4on:  
pUC  vector  

X-gal screening 17  
 2.  Phage  Vectors  (λ)  

• whether DNA ss or ds
• e.g. M13 ss; λ ds
• size of donor DNA
• less than 1-2 kb in M13
• upto 15 kb in λ 18  
 3.  Cosmid  Vectors  
• For  the  cloning  of  large  inserts  
– 35  –  45  kb  inserts  
• Hybrids  of  phage  λ  and  bacterial  plasmid  
– composed  of  the  cos  site  of  phage  λ  inserted  into  a  plasmid  
– cos  sites  are  necessary  for  packaging  of  phage  DNA  into  phage  
par-cles  
– λ  used  as  a  “syringe”  to  insert  the  insert  into  E.coli  
• Once  inside  the  bacterial  cell,  cosmids  replicate  as  plasmids  

19  
An  Example  of  a  Cosmid  Vector  

20  
 4.  Bacterial  Ar-ficial  Chromosomes  
(BACs)  
• To  clone  large  fragments  of  DNA  
• BACs  are  large,  but  low  copy  number  (only  1-­‐2  copies/  cell)  
• BACs  are  based  on  F  factor  
• Inserts  can  be  100  –  300  kb  
• Contains  
– origin  of  replica-on  
– selectable  marker  gene  
– mul-ple  cloning  sites  

21  
5.  Yeast  Ar-ficial  Chromosomes  (YACs)  
• YACs  can  be  used  to  express  eukaryo-c  proteins  that  require  
post-­‐transla-onal  modifica-on  
• Inserts  can  be  of  several  100  kb  (up  to  2000  kb)  
• Disadvantages:    
– less  stable  than  BAC  
– producing  "chimeric  effects”    
– ar-facts  where  the  sequence  of  the  cloned  DNA  actually  
corresponds  not  to  a  single  genomic  region  but  to  mul-ple  
regions.    
• chimerism  due  to  co-­‐liga-on  of  mul-ple  genomic  segments  into  
a  single  YAC  or  recombina-on  of  two  or  more  YACs  transformed  
in  the  same  host  Yeast  cell   22  
An  Example  of  YAC  

23  
 6.  Ti  Vector  for  Plant  Cells  
• Agrobacterium  tumefaciens  (now  known  as  Rhizobium  
radiobacter)  can  be  used  to  transform  plant  cells  with  T-­‐DNA  
containing  foreign  DNA  
• The  T-­‐DNA  and  insert  integrate  into  the  plant  genome    
• The  plant  cells  can  be  grown  in  -ssue  culture  and  eventually  
regenerate  a  mature  plant  carrying  a  foreign  gene  

Animation
24  
 Cloning  Vectors  
Vector   Form  of  DNA   Host   Capacity   Uses  
Plasmid   Circular   E.  coli   <15  kb   subcloning  &  
cDNA  libraries  
Lambda   Linear  phage   E.  coli   <23  kb   cDNA  &  
chromosome   genomic  
libraries  
Cosmid   Circular   E.  coli   30-­‐45  kb   Genomic  
  libraries  
BAC   Bacterial   E.  coli   100-­‐300  kb   Genomic  
chromosome     libraries  
 
YAC   Yeast   S.  cerevisiae   250-­‐2000  kb   Genomic  
chromosome   libraries  
 

25  
Replica-ng  Recombinant  Molecules  
• Need  many  copies  in  purified  form  
• In  general,  there  are  2  ways  to  produce  large  quan--es  of  
single  recombinant  molecule:  
– chemically  
• selec-vely  replicate  recombinant  DNA  molecule  in  a  test  tube  
• e.g.  PCR  
– biologically  
• selec-ve  replica-on  machinery  of  bacterial  or  simple  eukaryo-c  
cells  to  do  the  job  for  us  by  “tricking”  those  cells  into  replica-ng  
a  recombinant  DNA  molecule  of  interest  
   
26  
 Polymerase  Chain  Reac-on  (PCR)  
• Developed  by  Kary  Mullis  in  1986  (Nobel  Prize  in  1993)  
• PCR  copies  a  specific  DNA  sequence  through  in  vitro  
reac-ons  that  can  amplify  target  DNA  sequences  
present  in  very  small  quan--es  
• Principle:  DNA  made  in  one  amplifica-on  cycle  is  used  
as  template  in  subsequent  cycle  
– heat  denatura-on  to  yield  single-­‐stranded  DNA  
– annealing  of  primers  (oligonucleo-des)  to  single-­‐stranded  
DNA  
– extension  of  primers  by  thermostable  DNA  polymerase  
• Highly  sensi-ve,  requiring  as  licle  as  one  copy  of  single-­‐
stranded  DNA  as  ini-al  template   27  
 Polymerase  Chain  Reac-on  (PCR)  
• PCR  requires  two  oligonucleo-de  primers    
- one  complementary  to  the  3'  end  of  one  strand  of  the  DNA  to  
be  amplified  
- other  complementary  to  the  3'  end  of  the  other  strand  

• The  primers  anneal  to  denatured  DNA,  and  the  

complementary  strands  are  synthesized  by  a  heat-­‐stable  
DNA  polymerase  in  the  presence  of  Mg2+  and  the  four  
dNTP  
• The  three  steps  of  PCR—denatura-on,  primer  
annealing,  and  extension—are  repeated  over  and  over  
using  a  thermocycler  to  amplify  the  DNA  exponen-ally  
28  
Polymerase  Chain  Reac-on  (PCR)  

29  
 Limita-ons  of  PCR  

• Informa-on  of  the  target  must  be  known  to  synthesize  

primers  
• Even  minor  contamina-on  is  a  major  problem  
• Can  not  amplify  long  segments  of  DNA  

30  
 Applica-ons  of  PCR  
• Primers  can  be  designed  to  dis-nguish  1  bp  difference  in  
targets  
– detect  muta-ons,  gene-c  disorders  
• Important  for  studying  samples  from  single  cells  
– fossils,  crime  scenes  
• Can  explore  uncharacterized  DNA  regions  adjacent  to  known  
regions  
• RT-­‐PCR  is  a  powerful  tool  for  studying  gene  expression  
• Quan-ta-ve  real-­‐-me  PCR  (qPCR)    
– powerful  and  rapid  technique  in  quan-ta-vely  measuring  
gene  expression   31  
Selec-ve  Replica-on  inside  a  Host  Cell  
• Requires  use  of  cloning  vector  or  ar-ficial  chromosome  
• Recombinant  molecule  must  enter  host  cell  
• Recombinant  molecule  must  efficiently  replicate  
• Replicated  recombinant  molecule  must  be  recovered  from  
host  clone  
• Many  commercial  products  available  
• Transforma-on  into  expression  system  
– bacterial  cell,  e.g.,  E.  coli  
– eukaryo-c  cell,  e.g.,  yeast  

32  
33  
 Host  Cells  for  Cloning  Vectors  
• Widely  used  prokaryo-c  host:  E.  coli  
• Widely  used  eukaryo-c  host:  Saccharomyces  cerevisiae  
– Why?  
– it  can  be  grown  easily  
– its  gene-cs  have  been  studied  intensively  
– its  genome  has  been  sequenced  
– it  can  poscransla-onally  modify  eukaryo-c  proteins  
– it  is  considered  to  be  safe  

34  
35  
 Amplifica-on  and  Recovery  
• Introducing  Foreign  DNA  into  Cell  
1. transforma-on  for  plasmids  
• Ca2+  and  heat-­‐shock  
• electropora-on  
2. transduc-on  for  phages  
• forma-on  of  recombinant  plasmid/chromosome  
• produc-on  of  progeny  phages  carrying  the  recombinant  DNA  molecule  

3. gene  gun  (aka  bioblaster)  

4. microinjec-on  into  the  nucleus   36  
Gene  Gun   Microinjec-on  

37  
 Amplifica-on  and  Recovery  Cont.  
• Replica-on  of  vector  and  insert  u-lizing  host  cell  machinery  
– presence  of  an  origin  of  DNA  replica-on  
– exist  as  closed,  circular  ds  DNA  molecule  
 
• Recovery  of  recombinant  DNA  
– purifica-on  of  bacteriophages  
• chemical  extrac-on  
– purifica-on  of  extra  chromosomal  plasmids  or  recomb.  
molecules  
•  chemical  or  mechanical  extrac-on  
• centrifuga-on  and/or  chromatography  for  plasmids   38  
 Recombinant  Libraries  
• Collec-ons  of  cloned  sequences  
• Ideally,  a  genomic  library  contains  at  least  one  copy  of  all  
the  sequences  in  the  genome  of  interest  
• Genomic  libraries  are  constructed  by  cusng  genomic  DNA  
with  a  restric-on  enzyme  and  liga-ng  the  fragments  into  
vectors  
• The  choice  of  vector  usually  depends  on  the  size  of  the  
genome  
• A  cDNA  library  contains  complementary  DNA  copies  made  
from  the  mRNAs  present  in  a  cell  popula-on  and  represents  
the  genes  that  are  transcrip-onally  ac-ve  at  the  -me  the  
cells  were  collected  for  mRNA  isola-on   39  
 cDNA  Library  
• A  cDNA  library  is  prepared  by:  
– isola-ng  mRNA  from  cells  
– synthesizing  the  single  stranded  complementary  DNA  using  
reverse  transcriptase  
– then  using  RT-­‐PCR  to  copy  the  single-­‐stranded  DNA  into  
double-­‐stranded  DNA  
– cloning  the  cDNA  molecules  into  a  vector  

40  
 Genomic  and  cDNA  libraries  
• Consist  of  collec-ons  of  DNA  molecules  
• Genomic  library  consists  of  fragments  of  genome  
– oten  constructed  from  par-al  restric-on  digests  
– typically  mul-fold  representa-on  of  inserts  
– contain  introns  and  regulatory  sequences  
• cDNA  libraries  consist  of  DNA  derived  from  mRNA  
popula-on  of  cell  types  or  -ssue  
– limited  to  transcribed  genes  
• not  all  mRNAs  are  expressed  at  any  given  -me  
– introns  and  flanking  regulatory  sequences  absent  
41  
 Iden-fying  DNA  Molecules  
• It  is  oten  a  challenge  to  iden-fy  desired  gene  in  
library  of  thousands  of  clones  
• Using  nucleo-de  probes  
– principle  based  on  base-­‐pair  complementarity  
– colonies  or  phage  plaques  are  transferred  to  
membrane,  lysed  and  DNA  is  denatured  
– probe  is  applied  to  membrane  
• labeled  with  radioac-ve  isotope  or  fluorescent  dye  
• probe  forms  double  helix  with  complementary  DNA  
– hybrid  DNA  is  iden-fied  in  autoradiogram  or  by  
exposure  to  exci-ng  wavelength  of  light  
42  
43  
 Reference  
• Chapter  20  &  22  from  Concepts  of  Gene-cs,  10th  Edi-on  
(2012)  by  Klug,  Cummings,  Spencer  and  Palladino  
• Chapter  17  from  Gene-cs:  an  integrated  approach  
• Chapter  8  from  Modern  Gene-c  Analysis  

44  
 Gene-c  Engineering  
• Gene-c  engineering  is  where  recombinant  DNA  (rDNA)  
technology  is  used  to  introduce  desirable  traits  into  
organisms  -­‐-­‐>  alter  genotype  of  organism  
• A  gene-cally  engineered  (GE)  plant/  animal  contains  a  rDNA  
construct  producing  a  new  trait      
• Conven-onal  breeding  methods  have  long  been  used  to  
produce  desirable  traits  in  plants/animals,  GE  is  a  much  more  
targeted  method    
• Engineered  genes  (transgenes)  are  used  to  construct  
transgenic  organisms  
• Numerous  applica-ons  in  addi-on  to  study  of  genes   45  
 Applica-ons  of  Gene-c  Engineering  
1. Medicine  &  Healthcare  
– produc-on  of  hormones,  biochemicals,  vaccines  
2. Molecular  biology/  research  
3. Diagnos-cs  
4. Agriculture  
– golden  rice  
– herbicide-­‐resistant  crops  (Roundup™)  
– insect-­‐resistant  crops  (Bt  toxin)  
5. Animals  &  Humans  

46  
 GE  in  Medicine  
• Produc-on  of  human  insulin  in  E.coli  (humulin)  

Discovery of insulin –
Nobel prize in 1923

47  
 GE  in  Medicine  
• Produc-on  of  human  insulin  in  E.coli  (humulin)  
• Produc-on  of  angiosta-n  and  endosta-n  (blocks  the  growth  
of  new  blood  vessels)  in  Pichia  Pastoris    (yeast)    
• Factor  VIII  &  Factor  IX  –  produced  in  cell  culture  for  the  
treatment  of  Haemophilia  A  and  B,  respec-vely  
• Erythropoie-n  (EPO)  –  produced  in  cell  culture  for  the  
treatment  of  anaemia  
• Hormones:    
– HGH  in  E.coli  
– human  parathyroid  hormone  in  E.coli  
– Oxytocin  in  E.coli  

48  
49  
Self-­‐Replica-ng  Synthe-c  Microbes  
• Announcement  of  synthe-c  bacteria  on  May  20,  2010  
(Gibson  DG  et  al.  "Crea-on  of  a  Bacterial  Cell  Controlled  by  a  Chemically  
Synthesized  Genome"  Science  Express  20  May  2010;  pp.  1-­‐7)  

– Mycoplasma  genitalium  with  only  485  genes  (2008)  

– minimal  genome  required  for  vitality  
– synthesized  1.08  million  base  pair  chromosome  of  a  
modified  Mycoplasma  mycoides  genome  
– ordered  gene  sequences  
– assembled  in  yeast  
– transferred  to  Mycoplasma  capricolum    
Photo Source: http://www.dailymail.co.uk/sciencetech/article-1279988/Artificial-life-created-Craig-Venter--wipe-humanity.html
 Fig. 5 Images of M. mycoides JCVI-syn1.0 and Wild type M.mycoides

D G Gibson et al. Science 2010;329:52-56

Published by AAAS
 Gene-c  Engineering:  Plants  
• Considerable  agricultural  importance  
• Considerable  controversy  regarding  health  and  
environmental  safety  
– recombinant  plants  oten  referred  to  as  GMOs,  
gene-cally  modified  organisms  
– argument  that  long-­‐term  effects  are  unknown  

• Advantages  &  Disadvantages  of  GM  food?  

53  
 Gene-cally  Modified  (GM)  Food  

Desirable  Characteris4cs  (+)   Undesirable  Characteris4cs  (-­‐)  

Feeding  7.2  billion  people  on  earth   Poten-al  risk  to  human  health?  -­‐-­‐>  
-­‐  worldwide  grain  produc-on      by  260%  from   increased  obesity  and  type  II  diabetes  in  last  
1950  to  1990   50  years?  

Disease/  frost/  draught  resistant  crops  -­‐-­‐>   Ecological  disrup-on  by  hybridizing  with  
less  chemical  pes-cide  use  -­‐-­‐>  less   na-ve  plants  (gene-c  drit  or  gene-c  
environmental  damage  -­‐-­‐>  less  pollu-on  in   trespass)  
rivers  &  streams  
Nutri-onally  enhanced  food  (golden  rice)  &   Developing  countries  are  made  dependent  
edible  vaccines  –  bananas  or  potatoes   on  developed  countries’  mul-na-onal  
containing  vaccines   companies    

Ripen  slower  and  last  longer  during  shipping   Unintended  harm  to  other  organisms  
-­‐-­‐>  less  food  wastage  
54  
 Gene-c  Engineering:  Plants  
• Two  major  methods  for  transforma-on  
1. Ti  plasmid  from  Agrobacterium  tumefaciens  
• upon  infec-on  of  plant  with  bacteria  containing  recombinant  
Ti  plasmids,  the  Ti  plasmids  are  transferred  and  inserted  into  
host  plant  genome  

• plasmid  itself  is  gene-cally  modified  to  include  polylinker  

(mul-ple  restric-on  sites)  and  drug  resistance  genes  

2. gene  gun  to  inject  DNA-­‐coated  micropellets  into  cells  

55  
Infec-on  by  A.  tumefaciens  

56  
 E.g.  Transgenic  Tobacco  Plants  

• Transgenic  Tobacco  Detoxifies  TNT  (trinitrotoluene)  

– serve  as  a  potent  detoxifier  when  equipped  with  a  bacterial  enzyme  nitroreductase  (NR)  
– hcp://www.scien-ficamerican.com/ar-cle.cfm?id=transgenic-­‐tobacco-­‐detoxi  

• Other  uses  include  produc-on  of  collagen,  interleukin,  PHB,  cellulase  

57  
 Pharmacogenomics  
• Designer  drugs  
• Influence  of  gene-c  
varia-on  on  drug  
response  in  pa-ents  
by  correla-ng  gene  
expression  with  a  
drug's  efficacy  or  
toxicity  

58  
 Gene-c  Engineering:  Animals  
• Numerous  model  systems  and  applica-ons  
– Three  methods  of  crea-on  of  transgenic  animals:  
1. DNA  microinjec-on  
2. Retrovirus-­‐mediated  gene  transfer  
3. Embryonic  stem  cell-­‐mediated  gene  transfer  
 

DNA microinjection Retrovirus-mediated gene transfer 59  

Embryonic  Stem  Cell-­‐Mediated  Gene  
Transfer  
 

60  
 Gene-c  Engineering:  Animals  
• Uses  
– ANDi:  the  first  transgenic  Rhesus  monkey  with  gfp  gene  
– pets  (GloFish)  –  may  be  modified  to  be  used  for  pollu-on  
detec-on?  
– research  –  understanding  specific  gene  func-on  (fruit  flies)  
– reducing  pollu-on  (Enviropig  excretes  from  30  to  70%  less  
phosphorus  in  manure)  
– dairy  cows  producing  human  breast  milk    

ANDi Glo Fish 61  

 Cloning  of  Dolly  

Dolly was the first mammal to be

cloned from an adult cell

The project showed that adult DNA, which

has become fixed to do a particular job in its
cell, can be reprogrammed to create an
entirely new organism
62  
 Gene-c  Engineering:  Humans  
• Gene  replacement  therapy  or  gene  therapy  
– curing  the  abnormal  condi-on  by  introducing  
transgenic  DNA  from  wild  type  alleles  

• Many  technical  and  ethical  issues  

– implica-ons  for  gene  pool  for  germ-­‐line  gene  therapy  
– what  traits  cons-tute  disease  rather  than  just  a  
characteris-c  
– risk  versus  benefit  

63  
Types  of  Gene  Therapy  in  Mammals  

64  
 Soma-c  Cell  Human  Gene  Therapy    
• in  clinical  trials  
• First  case  -­‐  A  4  year  old  girl  in  1990  in  US  
– adenosine  deaminase  (ADA)  deficiency,  a  gene-c  disease  à  
severe  combined  immunodeficiency  disease  (SCID)  

65  
 Risks  of  Human  Gene  Therapy  
• 18-­‐year-­‐old  American  (Jesse  Gelsinger)  became  the  first  
person  to  die  from  gene-­‐therapy  research  
– ornithine  transcarbamoylase  (OTC)  deficiency,  a  metabolic  
disorder  à  problem  with  elimina-on  of  ammonia  
– Jesse  had  par-al  OTC  deficiency  à  manage  with  diet  &  meds  
– signed  up  for  human  gene  therapy  trials  at  U  of  Pennsylvania  
– Sept.  13  ’99  -­‐  infusion  of  correc-ve  OTC  gene  encased  in    
acenuated  recombinant  adenoviral  vector  
– he  experienced  a  severe  immune  reac-on  to  the  vector  à  
died  4  days  later  
• Three  children  died  in  June  2020  from  gene  therapy  for  their  
rare  muscle  disease  
 Germ-­‐Line  Human  Gene  Therapy  

• CRISPR Cas9 invented by Jennifer Doudna & Emmanuelle Charpentier in 2013

---> Nobel prize in Chemistry in 2020
 Ethics  in  Gene-c  Engineering  
• Latest  news:  2018  –  has  designer  babies  arrived???  
– Chinese  researcher  He  Jiankui  claimed  making  the  world’s  first  
gene4cally  edited  babies  using  CRISPR  
– the  research  isn’t  published  in  a  peer-­‐reviewed  journal  à  
successful  edi-ng  remain  to  be  unverified  
– via  IVF  –  males  had  HIV  à  edited  to  disable  HIV  protein  
–  produced  22  embryos  using  IVF  à  HIV  protein  was  disabled  in  
16  embryos  à  11  embryos  implanted  in  6  acempts  à  one  
successful  pregnancy  à  twin  girls  
– one  baby  has  both  copies  of  the  altered  gene  à  likely  unable  to  
contract  HIV  but  other  baby  has  only  one  copy  
68  
• similar  to  vaccina-ng  to  protect  against  an  infec-ous  
disease  or  a  gene-c  advantage?   69  
 Ethics  in  Gene-c  Engineering  
• Use  of  human  embryos  for  gene-c  manipula-on  –  illegal  in  
Canada  
• Do  we  have  the  right  to  alter  the  gene-c  makeup  of  our  
descendants?  
• Removal  of  undesirable  traits  (such  as  avoidance  of  gene-c  
condi-ons)  vs.  inser-ng  desirable  traits  (or  enhancements)  
–  preven-on  from  being  suscep-ble  to  HIV  vs.  enhancement  
with  higher  IQ  or  athle-c  build  

• Crea-on  of  eugenics?  

70  
 Reference  
• Chapter  20  &  22  from  Concepts  of  Gene-cs,  10th  Edi-on  
(2012)  by  Klug,  Cummings,  Spencer  and  Palladino  
• Chapter  17  from  Gene-cs:  an  integrated  approach  
• Chapter  8  from  Modern  Gene-c  Analysis  

71  
 Addi-onal  Informa-on  
• Video:  How  to  use  a  gene  gun  
– hcps://youtu.be/gnz5zlzsRYw  

• Bacterial  Produc-on  of  Human  Insulin  

– hcp://care.diabetesjournals.org/content/4/1/64.full.pdf  

72