Basic transgenes
What are the problems associated with genetically modified bacteria
Gene and control sequences
Protein activity
Protein recovery
What is DNA Microinjection? and its Steps?
Microinjection of transgene into pronuclei or nucleus of a fertilized egg
First method used to make transgenic animals
Steps - Insertion of micropipette into pronucleus, inject transgene construct, embryo transferred to recipient, Detection of transgenics in live born offspring, Analysis of gene incorporation
What is embryonic stem cell mediated gene transfer
Culture the harvested ES cells from donor blastocyst
Transfect the transgene and select the transfected ES cells
Transgenics ES cells are incorporated into recipient blastocyst, places into surrogate
Maturing host animal is chimeric
some of their offspring may be transgenic
What is RNA interference
dsRNA processed into small interfering RNA
These associate with RNA silencing complexes that guide to target mRNA
Enzyme called "slicer" cleaves the target mRNA
What is CRISPR-Cas9
Clustered regularly interspaced short palindromic repeats - sequences discovered in the bacterial system to detect and defend against viruses
Cas - Small clusters of cas genes (CRISPR-associated system) are located next to CRISPR sequences
How are transgenic animals being produced?
Molecular biology combines techniques from genetics, cell biology, developmental biology and microbiology
How do you increase efficiency rates of gene integration in DNA microinjection
Decreasing the size of the microinjection needle
Concentration of DNA
Experienced people
Stage of egg development and quality
Embryonic stem cell insertion advantages and disadvantages
A = ESCs are easily modified and easy to grow, they can differentiate into any cells, gene transfer to ESCs can be used for studying developmental pathways
D = chimeras result - not transgenic animals, lack of suitable ES cell lines in livestock
What is morpholino knockdown
Morpholinos - synthesized oligonucleotides that block transition and/or splice sites
Stable to nucleases
Don't trigger an immune response
Are not incorporated into the genome
What are the steps of CRISPR
Single guide RNA = Sequence that is specific to the DNA target + tracrRNA
Cas9 + sgRNA bind
Complex binds to target sequence and result sequence and result in target-specific double-stranded DNA cleavage
The cleavage site will be repaired by the non-homologous end joining DNA repair pathway, an error-prone process that may result in insertions/deletions that may disrupt gene function OR if donor DNA with homologous sequence to the target is present, homologous directed repair will result in the incorporation of the new DNA
What are methods of making transgenic animals
Recombinant DNA
Genetic Cloning
Gene expression
Genomic mapping
CRISPR-Cas9
What is viral infection?
Retroviral infection
Retroviruses integrate by a defined mechanism into the genome of the infected cell - RNA reverse transcribed into DNA, dsDNA integrates into genome, make recombinant retroviruses that are replication defective
Remove non-essential viral genes, insert transgene construct
How does fusion of the vector to spermatozoa
Sperm as delivery
Mix with DNA with sperm
Intracytoplasmic sperm injection
Fertilization of egg
What is different between homologous recombination vs non-homologous end-joining
HR = genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA, insertion of gene, insertion of correction, repairing of double stranded breaks
nHRej = Repair through an error prone pathway that results in a deletion or insertion in target gene, requires no template
What are the unintended effects of transgenes
Off-target effects = genome editing can lead to unintended mutations which may have unknown implications
Ecological effects = ecosystem disruption
How do you construct a transgene
Redesign the gene for the altered host (i.e. remove introns)
Combine with a specific promoter
Attach enhancers (if required)
What are the advantages vs disadvantages of viral injection
Advantages - Technically easy, introduction into embryo can occur at various stages, up to 100% infected
Problems - Integration is random, viral DNA is introduced into the cell, mosaicism, breed many times to get homozygous transgene in every cell, size limitation to the gene
Knockdown vs Knockout?
Knockdown - inhibit/down regulate = Antisense RNA, gene shears, RNA interference, Morpholino
Knockout - remove/disrupt gene = Homologous recombination, non homologous end-joining
What are zinc finger nucleases
Binding domain recognizes, nucleotide triplets
DNA cleaving domain "molecular scissors"
Deletion or insertion disrupts the gene
Disadvantage - Not every triplet has a ZF nuclease associated with it, ZF sometimes interact with each other
Ethical issues with genome editing
Use in humans
Therapeutic or enhancement
Equity and access
moral and philosophical concerns - natural processes, cultural and religious views
Balance of risk and benefit
What are methods of delivery for transgenes
Microinjection
Viral infection
Embryonic stem cells or somatic cells
Sperm-mediated DNA transfer
How are transgene delivered through chemicals
Transgene can be delivered using natural or synthetic compounds to facilitate the transfer of genes into cells
Transfection is the process of introducing transgene into eukaryotic cells by nonviral methods
Pores in the animal cell are chemically opened which allow the transgene to enter the cell
What are antisense genes
Antisense genes are made by positioning a DNA fragment coding for the target RNA in the reverse orientation between a strong promoter and a termination signal
What are TALENs
Transcription activator-like effector nucleases
Binding domain recognizes single nucleotides
design the TALEN to bind to specific DNA sequence + cleaving domain
Higher specificity than ZF
What are some transgene examples in animals that we have seen
Tracy the sheep
Pig for human organs
transgenic chicken
Mastitis
Animal welfare cow with polled gene
Enviro pig