Lecture notes on transgenic animals (Based on 'Biotechnology' by David P. Clark, Nanette J. Pazdernik, second edition)

1. Transgenic Animals
Presenter:
Pramod Acharya
4/10/2018

2. Topics of Discussion
Day I
 New and Improved Animals
 Creating Transgenic Animals
 Larger Mice Illustrate Transgenic Technology
 Recombinant Protein Production using Transgenic Livestock
 Knockout Mice for Medical Research
 Alternative Ways to Make Transgenic Animals
 Location Effects on Expression of the Transgene
 Combating Location Effects on Transgene Expression
 Targeting the Transgene to a Specific Location
 Deliberate Control of Transgene Expression
 Inducible Endogenous Promoters
 Recombinant Promoter Systems
 Transgene Regulation via Steroids Receptors

3. Topics of Discussion
Day 2
 Gene Control by Site-Specific Recombination
 Transgenic Insects
 Practical Transgenic Animals
 Transgenic Animals Help Fight Disease
 Genetically Modified Mosquitoes
 Transgenic Animals Improve the Food Supply
 Other Uses for Transgenic Animals
 Applications of RNA Technology in Transgenics
 Natural Transgenics and DNA Ingestion

4. New and Improved Animals
 Understanding genetics: gives more idea for improving livestock and crops
 Novel genetic information inserted in germline, passed to descendants
 Transgenes: same species, closely related or unrelated species
 Tried in cats, sheep, dogs, even monkeys

5. Creating Transgenic Animals
 Transgene injected in fertilized egg cells
 Before fusing of two pronuclei (male and female),
DNA is injected in male pronucleus
 Success rate 5-40%
 Fertilized egg kept in culture during early divisions
of embryonic development
 Engineered embryos implanted in foster mother
 Founder animals (in which transgene is stably
integrated) allowed to mate to carry 2 copies of
transgenes
 In such mating, two copies (25%), no copies (25%)
and one copy (50%)
https://www.youtube.com/watch?v=i2t6ENxNoNU

6. Creating Transgenic Animals (Contd.)
 DNA integration may not be accurate
 DNA integration: more or less immediately
 Chimeric animals:
Only some cells contains transgenes (incomplete transgenesis)
 DNA integration is by random chances

7. Larger Mice Illustrate Transgenic Technology
 Somatotropin: growth hormone, single polypeptide encoded by single gene
 In 1982, somatotropin gene cloned and inserted into fertilized mouse eggs
 Genetically engineered mice were larger (almost double the normal size)
 Unrelated mouse gene, metallothionein was used a promoter
 Somatotropin manufacture in liver
instead of pituitary gland, size was double
 Recombinant human somatotropin (rHST)
 Dwarfism: defects in production
of somatotropin or shortage of receptors
 rHST used to treat hormone-deficient type of dwarf

8. Larger Mice Illustrate Transgenic Technology (Contd.)
Trendy transgenic mice
 Marathon mouse:
Run 1800 meters without exhaustion
 Mighty mouse:
Lacks myostanin (protein that slows
muscle growth)
 Fierce mouse:
NR2E1 gene deleted, shows abnormal aggression
 Smart mouse:
Has extra copy of NR2B gene, made to have
improved learning and memory

9. Recombinant Protein Production using Transgenic Livestock
Recombinant bovine somatotropin (rBST)
 Cloned somatotropin gene from cows expressed in bacteria
 This produced large amount of rBST hormone
 rBST is used to increase milk production
 Hormone injection does not produce giant cows
Human insulin
 E. Coli are cultured to make recombinant proteins (expensive method)
 Use of livestock to express this proteins makes the product cheaper

10. Recombinant Protein Production using Transgenic Livestock
(contd.)
 Gene of interest inserted in place of β-casein gene
 Transgene expressed using endogenous promoter and 3’ regulatory elements that restrict
β-casein expression in goat milk
 10,000 quarts of milk per cow per year; enough processing & collection industries
 Gene expression only in mammary glands

11. Recombinant Protein Production using Transgenic Livestock
(contd.)
Recombinant Tissue Plasminogen Activator (rTPA)
 Small scale proteins production for clinical use
 Transgenic goats used
 rTPA: used for dissolving blood clots
ANDi
 Inserted DNA (read backwards)
 Successfully cloned rhesus monkey carrying gfp genes
 Used as a model

12. Knockout Mice for Medical Research
 Widely used on medical research to investigate gene
function
 They have selected genes inactivated “knock out”
 Used for genetic analysis of inherited diseases and cancer
Gene knock out process in mice:
1. Target gene is cloned
2. Gene is disrupted by inserting a DNA cassette into its
coding sequence
3. Inserted DNA segment prevents correct protein synthesis
4. Inactive gene copy is reinserted into animal
5. Incoming disrupted gene replaces original, functional
copy by homologous recombination
6. Founder mice are breed together with both copies to
produce knockout mice

13. Alternative Ways to Make Transgenic Animals
Use of Engineered Retroviruses:
 Retrovirus can infect embryonic cells, thereby promoting transgenesis
Advantages:
 Single copy of retrovirus and transgene integrated into genome
 Does not require skills of microinjection
 Partial transgenic tissue can be compared to normal tissue on same animal
Disadvantages:
 Retroviruses carry limited amounts of DNA
 Founder animals always chimeras (no precision insertion)
 Rarely used to create fully transgenic animal

14. Alternative Ways to Make Transgenic Animals
Embryonic Stem Cells:
 Are derived from blastocyst (early stage of embryo)
 It can retain ability to develop into any body tissue, including germline
Process:
 Embryonic stem cells cultured and DNA introduced
 Engineered embryonic stem cells inserted in central cavity of embryo at blastocyst
stage
 Genetic chimeras formed (some transgenic tissues and some normal)
 Chimeric founder animals mated with wild-type animal
 Expression of transgenes are indicated by coat/ fur color
 Male chimeras can father many offspring when crossed with wild type females

15. Alternative Ways to Make Transgenic Animals
Embryonic Stem Cells:

16. Location Effects on Expression of the Transgene
 Transgenes may express differently even if they are from same origin
 Level and pattern of expression varies mainly due to location of transgenes
 Expression affected by nearby regulatory elements
 Physical state of DNA
Region containing large amount of heterochromatin
DNA tightly packed or methylated
DNA covered with non-acetylated histones
 Position effects confirmation:
Extraction of non-expressed transgenes from transgenic animal
Extracted transgenes used to construct another line of transgenic animals
If some animals shows transgenic characters, location effect of transgenes is
confirmed

17. Location Effects on Expression of the Transgene

18. Location Effects on Expression of the Transgene
Combating Location Effects on Transgene Expression
(use of appropriate regulatory elements)
a. Enhancer sequences :
 These sequences become dominant by controlling over nearby genes or clusters
(regulatory sequences), thus providing position-independent expression

19. Location Effects on Expression of the Transgene (Contd.)
Combating Location Effects on Transgene Expression
(use of appropriate regulatory elements)
b. Insulator sequences (boundary elements):
 These sequence block the activity of other regulatory elements
 The transgenes are flanked by two insulator sequences by protecting effects of
other regulatory elements

20. Location Effects on Expression of the Transgene (Contd.)
Combating Location Effects on Transgene Expression
(use of appropriate regulatory elements)
c. Use of natural transgenes:
Most transgenes contain cDNA
They differ from wild-type version of gene
Most transgenes: under control of viral or artificial promotors
Such promotors are short and convenient to engineer
Full length natural eukaryotes are less prone to location effect
Yeast artificial chromosomes (YACs) used for inserting full-length
genes for humanized monoclonal anti-bodies into mice

21. Location Effects on Expression of the Transgene (Contd.)
Targeting the Transgene to a Specific Location
 Homologous recombination used rather than
DNA injection to insert transgene to a
particular location
 Some transgenes require specific location for
expression
 Sometimes, there is need of inserting original
sequences in altered version
 Gene targeting relies on homologous
recombination
 Special targeting vectors are designed to direct
the integration
 Inserted DNA is flanked by homologous
sequences
 Target vectors often linearized before
transforming for effective recombination

22. Deliberate Control of Transgene Expression
 High-level transgene expression is preferred for industrial protein production
 But, some proteins are toxic at high amounts
 Like, in establishing the line of transgenic animals, low expression is preferred
 So, for functional analysis, switch gene expression on or off needed
a. Inducible Endogenous Promotors:
 Natural promotors from the host animal that respond to certain stimuli
 For e.g., zinc used as natural promotors for inducing metallothionein promotor in
mice
 Next e.g., heat shock promotor from Drosophila Hsp70 gene used to drive
expression of transgenes
Drawbacks:
Level of induction low, sometimes significant expression even in absence of inducing
signal, often toxic side effects, signals taken up slowly sometimes

23. Deliberate Control of Transgene Expression (Contd.)
b. Recombinant Promotor System:
LacI control of a transgene
 Rous sarcoma virus (RSV) promotor
transcription of lacI gene
 When construct is expressed in a
transgenic animal, LacI protein is
produced
 LacI repressor binds to the operator
site upstream of the transgene and
blocks expression
 When inducer IPTG added, it binds to
LacI, which fall off the operator site
 The transgene is then transcribed
from the SV40 promotor

24. Deliberate Control of Transgene Expression (Contd.)
b. Recombinant Promotor System:
Hybrid TetR-VP16 Transactivator system
 A hybrid protein made from TetR and
VP16 controls transcription of a
transgene
 The hybrid protein binds to a TetR
operator site, and VP16 domain
activates transcription
 When tetracycline is added, the TetR
domain binds this and is released from
the tetO site
 Tetracycline inhibits transgene
expression

25. Deliberate Control of Transgene Expression (Contd.)
b. Recombinant Promotor System:
Reverse TetR-VP16 Transactivator system
 The reverse TetR-VP16 hybrid protein
binds to tetO only when tetracycline is
present
 The transgene is expressed only in the
presence of tetracycline

26. Deliberate Control of Transgene Expression (Contd.)
c. Transgene Regulation via Steroid
Receptors:
 Some transgenic animals have the
transgene under the control of a
steroid-controlled promotor
 When the animal is treated with the
steroid, the steroid diffuses across
the cell membrane and binds to its
receptor in the cytoplasm
 The receptor-steroid complex then
enters the nucleus, where it binds to
the transgene promotor and turns
on the transcription

27. Any Questions?
https://www.youtube.com/watch?v=RzYhcXjksKc

28. Transgenic Animals
Presenter:
Pramod Acharya
4/12/2018

29. Topics of Discussion
Day 2
 Gene Control by Site-Specific Recombination
 Transgenic Insects
 Practical Transgenic Animals
 Transgenic Animals Help Fight Disease
 Genetically Modified Mosquitoes
 Transgenic Animals Improve the Food Supply
 Other Uses for Transgenic Animals
 Applications of RNA Technology in Transgenics
 Natural Transgenics and DNA Ingestion
 Latest Innovation and Researches About Transgenic Animals

30. Gene Control by Site-Specific Recombination
 After successful incorporation of transgene in germline chromosome, segments of
DNA are physically removed or inverted to achieve activation of transgene
 Recognition of short specific sequences by DNA-binding proteins
 Recombination between these two sequences
 Cre recombinase system from bacterial virus P1 recognize loxP
 Flp recombinase system from 2-micron plasmid of yeast recognize FRT
 Both recognize 34 base-pair sites consisting of 13 bp inverted repeats flanking a
central core of 8bp
Use of Cre/loxP system
 Removal of selective markers
 Activation of transgenes
 In vivo chromosome engineering
 Creation of conditional knockout mutants

31. Gene Control by Site-Specific Recombination
Site specific deletions in transgenic
animals
 Two loxP sites inserted into the
DNA, flanking the sequence to
be deleted
 When Cre recombinase is
activated, it recombines the two
loxP sites and deletes the
segment of DNA between them

32. Gene Control by Site-Specific Recombination
Two mouse Cre/lox Syetem
 One mouse has the target DNA
that is to be deleted flanked by
two loxP sites
 Second mouse has gene for
Cre recombinase under the
control of a tissue-specific or
inducible promotor
 When these mice will mate,
some of the offspring will
receive a copy of both genetic
constructs
 When the Cre protein is
induced, it will direct the
deletion of the target DNA
https://www.youtube.com/watch?v=oLPjiwM0G7A

33. Transgenic Insects
 P element transposons are found in insects, like Drosophila
which causes Hybrid dysgenesis
 Hybrid dysgenesis: High mutation rate that lowers the
proportion of viable offspring
 P-elements: flanked by 31 bp inverted repeats
 Any DNA sequence in between inverted repeats is transposed
 Engineered P-element inserted in susceptible insects to
introduce any DNA sequence

34. Transgenic Insects (Contd.)
 Two plasmids used: helper supplies
functional transposase, and carries
immobile P-element with deleted IR
 Vector plasmid: transgene + marker
flanked by P-element IR
 Both vectors injected in egg (posterior)
 Transposase is expressed resulting in
random transposition of transgene marker
 Some insertion occur in germ cell, mating
repeated to get some offspring with
successful insertion

35. Practical Transgenic Animals
Transgenic Animals Help Fight Disease
 Spreading of influenza
 Incorporation of lysozyme in cow’s milk to prevent from diarrhea
 Transgenic goats to produce antithrombin (blocks blood coagulation)

36. Practical Transgenic Animals (Contd.)
Genetically Modified Mosquitoes
 PiggyBac transposon from cabbage looper and Minos transposon from
Drosophila hydei widely used genetic markers
 Transgenes usually expressed from Drosophila promotors
https://www.youtube.com/watch?v=TnzcwTyr6cE

37. Practical Transgenic Animals (Contd.)
Transgenic Animals Improve the Food Supply
 Atlantic salmon
 More omaga-3 fatty acids in pigs and
sheep
 Remove allergens (β-lactoglobulin in cows
milk)
Other Uses for Transgenic Animals
 Enviropig™ (Ontario pig) – saliva degrades
phytate
 Transgenic silkworm to produce
fluorescent silk
 Zebrafish – green fluorescent protein
 MouSensor – olfactory bulb express
receptors for an explosive

38. Applications of RNA Technology in Transgenics
 Antisense RNA, Ribozymes and RNA interference used
 Insert an antisense gene (inverting the DNA sequence)
 cDNA library used in higher organisms
 That antisense gene transcribed to give antisense RNA, then after binding
with mRNA of corresponding sense gene
 Result: inhibition of gene expression (95%)
 Insert transgenic constructs that express ribozymes
 Ribozymes may be designed to cleave the mRNA
 Reduce expression

39. Natural Transgenics and DNA Ingestion
 Human genome contains bacterial origin
 Transmitted by retrovirus
 Natural transgenesis
Pathways for natural acquisition:
Viral transduction
Direct intake of DNA
 Short fragments of DNA from diet can be taken up and incorporated into
the host chromosomes
 Ingested DNA may cross the placenta to fetuses and newborns

40. Summary
 Nuclear microinjection, use of retroviruses, embryonic stem cells
 Transgenic animals: novel or improved capabilities or inactivated genes
 RNA-based methods may be alternative approach
 Location of transgene affects the transgene expression
 Modifications foe positional effects: provide specific regulatory sequences
for transgene
 Use of Cre or Flp recombinases to rearrange DNA segments
 Transgenic animals for improved human nutrition, monitor pollution,
make novel materials, eradicate diseases

41. Innovation and Researches About Transgenic Animals
Milk from transgenic goat expressing human lysozyme for recovery and
treatment of gastrointestinal pathogens

42. Innovation and Researches About Transgenic Animals
Expression of recombinant human lysozyme in bacterial artificial
chromosome transgenic mice promotes the growth of Bifidobacterium and
inhibits the growth of Salmonella in the intestine
 Generated a transgenic mouse model by a modified bacterial artificial
chromosome containing a recombinant human lysozyme.
 rhLZ was expressed in various tissues but not in milk.
 Bifidobacteria were significantly increased, whereas Salmonella were
significantly decreased in transgenic mice compared to wild type mice.

43. Innovation and Researches About Transgenic Animals
Animal models for evaluation of albumin-based therapeutics
 FcRn binds albumin and extends its serum half-life to 3 weeks in humans.
 Albumin is utilized to extend the serum half-life of short-lived
therapeutics.
 Species-specific binding of albumin to FcRn challenges preclinical
development.
 Humanized FcRn transgenic mice may serve as improved models.

44. Innovation and Researches About Transgenic Animals
Behavioral defects in a DCTN1G71A transgenic mouse model of Perry
syndrome
 Perry syndrome is a rare neurodegenerative disease characterized by
parkinsonism, depression/apathy, weight loss, and central hypo-
ventilation.
 Single mutation in the DCTN1 gene recapitulates symptoms of Perry
syndrome patients, and provides evidence that DCTN1G71A transgenic
mice represent a novel rodent model of Perry syndrome

45. Any Questions?
Thank you
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