Table of Contents

1. Introduction
2. ——— Lecture 1 ———
3. L1: History and Application of Transgenesis
   1. Mendelian Genetics → → → Genetic Information is DNA
   2. Tools for DNA Manipulation
      1. Material
      2. Enzymes
      3. Technique
   3. Getting Foreign DNA into Organisms
   4. Application of Transgenesis
      1. Suppl. Lesson #1 pt. 1
      2. Suppl. Lesson #1 pt. 2
      3. Suppl. Lesson #1 pt. 3
      4. Suppl. Lesson #1 pt. 4
4. L2: Construction of a Transgene
   1. Design of a Transgenic Construct
      1. Regions: Promoter
      2. Regions: Insulator
      3. Regions: Intron
      4. Regions: Poly-A Tail
5. ——— Lecture 2 ———
6. Tutorial
   1. Introduction — Casein Paper
   2. Results and Discussion
7. L3: Design of a Transgenic Construct Cont.
   1. Coding Sequence (CDS)
   2. Other UTRs
   3. Plasmid Construction
   4. Supplement Lesson 2
8. L4: Transgene Transfection
   1. Chemical-Based
   2. Physical-Based
   3. Particle-Based
9. L5: Transgene Integration & Gene Editing
   1. Double Strand Break Repair
   2. Transposons
      1. Class I — RNA Retrotransposon
      2. Class II — DNA Transposons
   3. Paper Proof-of-Concept
10. ——— Lecture 2 ———
11. L5: Gene Editing cont.
    1. Viral Vectors
       1. Adenovirus
       2. AAV
       3. Retrovirus/Lentivirus
12. L6: Induced Gene Expression
    1. Site-Specific Recombination
       1. Tyrosine Recombinases
       2. Serine Recombinases
       3. Applications
    2. Tet System
       1. Tet-OFF
       2. Tet-ON
       3. Advantage/Disadvantage
13. Gene Editing — Endonucleases
    1. Zinc Finger Proteins
    2. TALENs
    3. CRISPR-Cas

Introduction

——— Lecture 1 ———

1. History and Applications
2. Construction of a Transgene
3. Introduction of the Transgene
4. Transgene Integration and Gene Addition
5. Strategies for Transgenic Animal Creation
6. Preservation of a Transgenic Line

L1: History and Application of Transgenesis

What had to happen?

1. Understand that DNA is the genetic information.
2. Discovery of tools for DNA manipulation
3. Foreign DNA into organisms (animals)

Mendelian Genetics → → → Genetic Information is DNA

Grigor Mendel crossed pea species with different phenotypic character. He postulated that there are two factors of inheritance.

• Work forgotten for 34 years. → Rediscovered by Hugo de Vries, renamed genes. But what is the material?

Thomas Morgan and Nettie Stevens show that genes are carried by chromosomes by work in Drosophila.

• Dominant gene carried on the X chromosome, thus supporting that the driving factor of “genes” is genetic (as sex characteristics are known to be linked to chromosomes). But what part of the chromosome?

MacLeod and McCarthy performed work which used horizontal gene transfer between virulent and non-virulent bacteria, and heat-killing the virulent one to demonstrate that the gene was arising the virulent phenotype. Not enough proof, though.

Tatum and Beadle proved then that one gene codes for one protein using the mold Neurospora crassa. An enzyme which allows for survival on minimal media. After UV exposure, the mutants could no longer survive on the MM, so they cultured them on MM with suppl. amino acids. Some mutants could survive on just one media, leading to the conclusion that one event leads to the decrease in viability, thus one gene is responsible for one protein.

Hershey and Chase used radiolabelled phosphorous and sulphur to distinguish DNA and proteins. Using bacteriophages, they introduced the radiolabels into/onto cells, and saw that the sulphur is washed away during centrifugation, but the phosphorous is not.

Watson and Crick deduce that DNA is a double helix based on work by Rosalind Franklin.

Tools for DNA Manipulation

Material

Plasmids were discovered in 1940. Small transmissible factors shared among bacteria.

• Fertility factor in E. coli was the sharing of an antibiotic resistance gene between bacteria. E. Lederberg.

1952 → Plasmid defined as any extrachromosomal hereditary element by J. Lederberg.

Enzymes

1967 → DNA Ligase, facilitates joining of DNA strands together. Weiss and Richardson purified the enzyme polynucleotide ligase from E. coli infected with T4 bacteriophage.

• E. coli DNA ligase (lig): create the phosphodiester by cleaving NAD, but doesn’t work on blunt-ended DNA.
• T4 ligase: Uses ATP, and can ligate blunt-end DNA, as well as RNA and RNA-DNA chimera. Best at 37ºC
• Thermophilic bacterium DNA ligase: 48 hours at 65ºC, 1 hour at 95ºC.

1950-60 → Restriction enzymes found to cleave DNA creating either sticky or blunt ends.

• 1950 → Luria, Weigle, and Bertani found that E. coli bacteria infected by λ can alter or restrict the viral proliferation.
• 1960 → Type I restriction enzyme: works at any random points in the DNA.
  • SAM, ATP, and Magnesium as cofactors.
  • EcoRI, BamHI, HindIII, etc…
• 1970 → Type II restriction enzyme (HindIII) found by Smith, Kelly, and Wilcox. They recognize a consensus sequence for more specific cut sites.
  • Magnesium as cofactor.
• 1970 → DNA Mapping using restriction digestion.
• 1978 → Nobel Prize for Phys. and Med. awarded to Arber, Nathans, and Smith.

Other Types

• Type III: recognize non-palindromic sequences that are inversely oriented. Cutting 20-30 bases from recognition site.
  • Require SAM and Magnesium.
  • EcoP15 (originated from P15 plasmid)
• Type IV: recognize modified (such as methylated DNA)
  • McrBC from E. coli K-12.
• Type V: guided using RNAs to target specific palindromic sequences
  • CRISPR-Cas.

Technique

1977 → Sanger DNA Sequencing was developed.

• dNTPs and ddNTPs, fluorophores, etc…

1983 → Mullis developed PCR.

Getting Foreign DNA into Organisms

1973 → First artificial gene transfer between two living organisms. Chang and Cohen.

• E. coli and S. aureus.

1980 → Gordon et al. performed microinjection into pronuclei of fertilized mouse oocytes. The embryos were then implanted. Introduced DNA strain of simian virus 40.

1982 → Palmiter et al. introduced a DNA fragment containing rat growth hormone gene into mouse eggs.

• DNA fragment of mouse metallothionein-I promoter fused to structural gene of rat growth hormone introduced via microinjection. 21 mice developed, 7 of which carried the fusion, 6 of which grew significantly larger than the other mice.
• Klenow Fragment is a DNA Polymerase I fragment which retains 5→3 polymerase and 3→5 exonuclease activity, without 5→3 exonuclease activity, which can be used to repair sticky-ends.

Application of Transgenesis

Supplemental lesson document provided on eCampus.

Suppl. Lesson #1 pt. 1

1. The hypothesis of this article is that the immune suppression activity of T regulatory cells can be stimulated by transgenic ectopic expression of myelin basic protein (an autoantigen).
   1. Further, exploring liver, a tolerogenic organ, and its role in autoimmune suppression.
   2. Reduction of autoimmune reaction.
2. CRP-MBP is found only in the livers of the transgenic mice, but K5-MBP is found only in the skin and thymus of the transgenic mice. This shows that the expression is regulated differently between these two transgenes in different tissues of the mouse.
3. Experimental Autoimmune Encephalomyelitis caused by auto-immunization to MBP. Panels C and E illustrate that only the CRP-MBP and not the K5-MBP was able to reduce the incidence of autoimmune reaction in the mice (indicated by a lower score). Panels F and G show that both the vector and adenovirus methods of delivery are sufficient for the autoimmune suppression.
   1. Demonstrates that the auto-immunity to MBP is resolved by hepatic presence of MBP.

Suppl. Lesson #1 pt. 2

1. The relation between mucin and neutrophil elastase is that the MUC5 A5 mucin promoter is operated by NE, which is an effector of pathogenic antigens. Their hypothesis is that the ability of NE to activate the expression of promoters is dependent on presence of core regulatory sequences. From this information, you can deduce where these regions are.
2. They cut the promoter into different lengths upstream from the +48 start codon.
3. The modified promoter and the control are statistically significantly different from one another, indicating that

Suppl. Lesson #1 pt. 3

1. hDMD del45, mdx and mdxD2 were used to generate deficient Duchennes phenotypes because the LoF human variant of dystrophin is not sufficient to replace the absence of mouse dystrophin. mdxD2 has a more severe presentation of muscular dystrophy phenotype than mdx.
2. The authors used CRISPR/Cas9 so that the modification is done permanently do the genome
3. Compared to the “wild-type” hDMD mice.

Suppl. Lesson #1 pt. 4

1. The strategy of the authors to remove the IARS mutation is by reparation of the mutated codon with a synonymous codon for the correct amino acid in exon 3. To perform this, they performed a CRISPR/Cas9 guided cut in the exon 3 (E3) region of the gene, and inserted the repair as well as a GFP expression cassette flanked by a transposase recognition site. The gene was cloned and homologously recombined with the mutant allele at the E2 and E4 regions. The transformed fibroblasts were enriched via the GFP marker, then embryos were generated from these cells (via somatic cell nuclear transfer). Then, the GFP cassette was removed by introduction of piggyBac recombinase to create a repaired allele without GFP.
   1. SCNT is a process where an enucleated oocyte is merged with a somatic cell, such that the nucleus of the somatic cell is “reprogrammed” by the oocyte to act as its new nucleus.
2. To facilitate the removal of the expression cassette.
3. Restriction enzyme recognition sites (restriction sites) to produce known fragment sizes post digestion.
4. Using GFP to enrich the population, then take GFP negative cells. Performed by cell sorting.

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Gene inactivation is a common application of transgenesis.

A gene can also be modified without leaving a trace of foreign DNA.

L2: Construction of a Transgene

Design of a Transgenic Construct

Inclusion of extra sequences between the different elements is possible, and size doesn’t matter for processes other than vector efficiency.

Regions: Promoter

Region of DNA which leads to initiation of transcription of a gene.

~0.1-1 kb long.

Leads to recruitment of RNAP II.

Regions: Insulator

Prevent the SILENCING of euchromatin (prevent heterochromatin formation) even when surrounded by heterochromatin.

They can also prevent distal enhancers from acting on the promoter of neighbouring genes.

E.x. cHS4 in beta-globin locus in vertebrates (chicken β-globin)

Regions: Intron

Non-coding regions of a gene.

Intron-mediated element/enhancement → IMEs are generic name that describes the intron’s ability to regulate a gene.

May code for functional ncRNAs such as miRNA or piRNA.

May also prevent R-loops (mRNA hybridization to its DNA template)

Regions: Poly-A Tail

Long chain of A that is added to mRNA during RNA processing to increases the stability of the mRNA molecule. Promotes the exportation from the nucleus, recruitment to ribosomes, and inhibits degradation.

Also aids in proper termination of mRNA. Part of typical TU terminators (which would also include a loop for further prevention of transcription.

——— Lecture 2 ———

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Tutorial

Introduction — Casein Paper

1. Cow’s milk is consumed in many ways, so increasing not only nutritional value but also functional properties of the milk is beneficial for many people and industries.
2. 80% casein (for different isoforms), water and calcium phosphate which is solubilized by the casein micelles in colloids.
3. Casein is a large part (80%) of milk’s nutritional values. By increasing the level of casein, you can increase the nutrition of the milk. Specifically, beta to determine calcium level and kappa which regulates micelle size (and thus cheese capabilities).
4. The aim is to increase expression of beta and kappa casein in a cattle. Introducing additional copies of the beta and kappa casein genes into female bovine fibroblasts using nuclear transfer.

Results and Discussion

…

L3: Design of a Transgenic Construct Cont.

Coding Sequence (CDS)

Usually using the cDNA sequence, so that it is not too long (and so that you select the right isoform if there is splicing events).

Codon Optimization…… You get it…

• For animals, you might increase immunogenicity of the DNA by including too many CpG islands (which, when methylated, are antigens)
• Kudla et al.

Can add epitope tags for Ig recognition (V5, Myc, HA, Spot, T7, NE) and affinity tags (His, Strep, MBP, CBP) or chromatography tags (FLAG). You can also add fluorescent tags for visual readout (GFP, other FPs).

Other UTRs

Kozak Sequence: (gcc)gccRccAUGG

• R is ALWAYS a purine, AUGG is non-variable.
• Usually includes start codon in the non-variable region.

Plasmid Construction

If you cannot use an MCS (because you are not using good strategies), you can use TOPO-activated engineering of the plasmid, where a linearized plasmid is treated with topoisomerase, then added to PCR product which will reform a circular plasmid with an insert.

Supplement Lesson 2

1. Hypothesis: Expression of transgene products can be mediated/increased by the introduction of an intron to your construct.
2. Parts of construct:
   1. CMV promoter (cytomegalovirus, expressed everywhere/ubiquitous)
   2. IVS: synthetic intron (which does what?)
   3. Internal Ribosome Entry Site for polycistronic expression
   4. eGFP fluorescent reporter
   5. Other various introns (hCMVI, TPLI, SV40 (SVI), CHEF1)
   6. Poly-A

L4: Transgene Transfection

Very big and negative molecule. Not very good for diffusion through membranes.

Many techniques for transfection:

1. Chemical-based
2. Physical-based
3. Particle-based

Chemical-Based

Calcium Phosphate Method

• Combine HEPES and CaCl2 with DNA. The Ca2+ and phosphate will precipitate and bind DNA, forming small nanoparticles which are endocytosed.
• Heat shock can improve transfection rate.

Dendrimers

• Iron beads coated with dendrimers to form magnetic nanoparticles
• methyl propenyl ester with diaminoethane

Cationic Polymers

• Bound DEAE dextran or polyethylenimine to DNA
• Lipofection is where bilayer membrane of cationic lipids which will form a DNA colloid/complex.

Physical-Based

Needle Microinjection (1966)

• Small syringe to inject DNA, and a manipulator to hold the cell.
• Constant flow is simple, pulsed flow is more expensive but allows for control of amount into the cell (and avoids rupture).

Electroporation (1982; Neumann et al.)

• Electric field induced across the membrane which causes ingress of DNA into the cell.
• Able to be performed in a cuvette, as well as in whole animal/organism in targeted area.

Optical Transfection (1984; Tsukakoshi et al.)

• Create a hole with a laser!

Somatic/Protoplast Fusion (1986)

• Fusogenic agents such as Sendai virus to fuse cells where cell wall is removed.

Hydrodynamic Delivery (2005; Al-Dosari et al.)

• Efficient to transfect irrigated organs
• Pick an organ that has a clear and defined venous pathway, isolate the organ within that pathway, then super duper push liquid to increase pressure to force the gene into cells.
• Introduced by somewhere such as inferior vena cava

Sonoporation (2007; Song)

• High-intensity ultrasound to induce pore formation

Cell Squeezing (2013; Sharei et al.)

• Force cell through a capillary to constrict the shape of a cell, where the macromolecules are forced into the cell.

Particle-Based

Support to carry the transgenes.

Gene Gun (1987; Sanford et al.)

• Gold nanoparticles binding DNA, shot with helium to hope and pray to god that you shoot the particle into the nucleus.

Magnetofection (Plank et al.)

• DNA associated with (+)ve charged ferromagnetic beads, then use a magnetic field to force the particles into cells.

Impalefection (Mc Knight et al.)

• Force cell onto a nanofibre which impales the cells with the fibres.

L5: Transgene Integration & Gene Editing

Transient transfection vs*. Lentiviral transduction*

Double Strand Break Repair

Perform a double-strand break, add a sequence with homology recombination site, then you rely on the repair mechanism. Two mechanisms (homologous integration, and non-homologous end-joining).

Transposons

Replicative vs Conservative transposition mechanisms.

Transposition is mediated by an enzyme called a transposase which is able to cut the DNA strand and insert the DNA.

Class I — RNA Retrotransposon

Strong similarity to retroviruses (possible lineage of evolution)

Class II — DNA Transposons

DNA is cleaved and bound to the actual transposase and moved to a transposable element.

For gene transfer, usually using an exogenous transposase to trigger the transposition just once.

• Transposase can be delivered in a vector or just by mRNA injection.

Can use a “seed male” to cross with other mice to generate possible-to-screen strains of mice.

Sleeping Beauty or Piggy Bac are usually used for transgenesis (P element is naturally present in Drosophila, and Tol2 is also an option).

Each transposition system has its own integration tropism.

Paper Proof-of-Concept

Goodnotes notes.

——— Lecture 2 ———

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L5: Gene Editing cont.

Viral Vectors

DNA/RNA gotta get in there, protein coat, SOMETIMES a lipid envelope.

Which have been used in animal transgenesis or gene therapy

• Adenovirus
  • dsDNA, non-enveloped, non-integrating
  • 36kb genome, 30kb capacity, high immunogenicity and toxicity (lytic)
  • Broad host range, immunogenicity can be reduced
• Adenovirus + adenovirus associated virus (AAV)
  • ssDNA, no-enveloped, integrating (specific location)
  • 4.7kb genome, 4.5kb capacity, low immunogenicity or toxicity
  • Specific tissue tropism
• Retrovirus
  • (-)ssRNA, enveloped, integrating (random location)
  • 8.3kb genome, 8kb capacity, mid immunogenicity and very toxic
• Lentivirus
  • same but lower immune response and toxicity

Adenovirus

ADP - adenovirus death protein causes cell lysis

ITR - inverted terminal repeats, integration of viral DNA into host genome.

1. start with normal
2. remove replicative genes
3. remove all the genes and use space for transgene

AAV

Need presence of Adenovirus to be infectious. If alone, not pathogenic. Able to infect non-diving cells with no random integration.

Targeting specific types of tissues (different variants of AAV)

Retrovirus/Lentivirus

Lenti - HIV 1/2

Only infect dividing cells.

L6: Induced Gene Expression

Site-Specific Recombination

Allow two DNA strands to change their places (insertion, deletion, and inversion.

30-200 nt with partial inverted repeat symmetry.

Dre/Rox, Cre/Lox

Serine recombinase vs. Tyrosine recombinase

Tyrosine Recombinases

Most commonly used in transgenesis.

1. Cleave DNA strand, then form a crossed-strand intermediate

Serine Recombinases

Cut, rotate, re-ligate, dissociate

Applications

Stable lines to cross (Floxed mice)

Tet System

Originating from tetracycline resistance found in gram-negative bacteria.

• Transposon Tn10 has a tetR gene.
• Gene under control of an operon (tetO1 and tetO2 operators, AKA TRE)
• When TetR is bound to the operator, there is no expression.
  • Tetracycline binds to TetR, and derepresses the tetA channel protein.

Tet-_ means _ when tetracycline is present

• Tet-ON → expression with tetracycline;
• Tet-OFF → repression with tetracycline.

Tet-OFF

tTA gene is a fusion protein between TetR and VP16 — tetracycline-dependent transactivator.

• Repressor/activator fusion → activator function

Tet-ON

By random mutagenesis, found a reverse phenotype rtTA — reverse tetracycline-dependent transactivator.

Advantage/Disadvantage

Tetracycline is orthogonal to animal systems, but also can no longer use tetracycline in any experimental preparations.

Gene Editing — Endonucleases

Zinc Finger Proteins

ββα fold motif, Cys2His2 which binds

Cys2His2

• ββα fold
• Most characterized
• large variety of functions

Variants:

• Gag-knuckle
• Zinc ribbon
• Zn2/Cys6

N→C is 3’→5’

Each motif recognizes 3 bases (3 motifs for 9 is good)

How to select your ZFN for your specific function?

• Random selection using phage display to find a protein which is most suitable to bind the desired sequence.
• Phage Display, where the substrate is the desired binding sequence.
• FokI needs to dimerize to perform double strand break, so the binding sequences need to be up and downstream the cut site, facing the middle. Downstream on top strand, upstream on bottom strand (because 3’→5’ recognition

Zinc Finger Transcription Factors

• ZF-TF can be linked to VP64 activation domain (like in TetO)
• Also able to block TATA box to downregulate specific gene expression (by occupation)

TALENs

Transcription Activator-like Effector Nucleases → restriction enzymes which can cut DNA at specific sequences, as well.

Coupled with FokI (typically) as well. the TALENs have defined motifs which can select specific nucleotides by design. From Xanthomonas spp.

• Mostly conserved domain, except res.12-13 are variable, which can recognize specific nucleotides.

Used to create knock-out (non-homologous end-joining) or knock-in.

• Knockouts done in elegans, mice, rats, and hPSCs
• Knock-in don in cattle (against TB)
• Still somewhat used in 2025. 200x more expensive and very long to use……..

CRISPR-Cas

RNA-guided endonuclease Cas9 (CRISPR-associated protein 9).

If a bacteria survives a phage, they can “remember” it. They keep the sequences from invaders in the genome, which is located in the CRISPR region.

• Originally found in iap locus of E. coli
• Then Streptococcus thermophilus.
  • Some places with highly variable loci, with non-contiguous repeats separated by variable spacers.
  • Spacers contain bacterial memory of previous infections!
• tracrRNA is recognizing the repeated sequence between the spacers, so that the spacer sequences are liberated and can interfere with the phage genome.
  • The guide RNA is associated with the nuclease, so that it can recognize the viral genome and cut it.

Many types of Cas proteins.

• Can target ds or ss DNA
• Require or don’t the tracrRNA
• Number of nuclease domains
• Usually using spCas9 or saCas9 (strep or staph)
• PAM site variable

Next lesson online!