Presentation 8: Novel Transgenic Pigs Flashcards
What are Non-starch polysaccharides?
Barrier to nutrient hydrolysis and absorption
What are phytates?
Reduce nutrient digestibility and increased discharge
What is a transgenic animal?
The genome has been modified by the transfer of a gene(s) from one species to another
• Study gene function in disease
• Provide function reciprocal animals don’t have
How does somatic cell nuclear transfer work?
Donor animal, take cells and culture cells
1) Remove DNA from oocyte
2) Inject nucleus into enucleated egg cell and fusion
3) Egg implantation into host uterus
4) Cytoplast artificially activated
5) Only some of these cells will take transgene and be integrated
How were the transgenic pigs used in this study?
4 microbial genes added to genome
Co-expression of enzymes in saliva
Improve grain digestibility & nutrient emission
Increased energy levels & productivity and reduced pig feed
Reduce environmental impacts with decreased bodily waste level
How were the transgenes constructed?
The original signal peptide of genes were replaced by the signal peptide of the porcine parotid secretory protein (PSP).
Genes were synthesized using Gene script and then ligated into eukaryotic expression vectors
What is a monocistronic transgene?
Codes one gene per mRNA
One gene inserted into genome
Expression of only one microbial enzyme
What is a quadstrinoic trangene?
Polycistronic- codes multiple genes per mRNA
Quadstrinoc: Four genes inserted into genome necessary for gene
Expression of multiple microbial enzymes expression
Use of linkers
Requires promoter capable of replicating multiple genes
Quadstrinoic are polycistronic, but not all polycistronic are quadstrinoic
How was the LoxP system used?
Loxp system used to remove gfp marker from genome
GFP used to know where gene is located in pigs
How was the salivary gland specific transgene developed?
Fusion of multiple codon-optimized B-glucanase genes
Developed quasi-cistronic expression using mPSP (mouse parotid secretory protein) promoter
Fused with N-terminal porcine PSP signal peptides
How was POLYCISTRONIC CASSETTE developed?
Used linker peptides E2A,T2A and F2A
Attached to HA tag
Fusion enzymes were ligated downstream of the CMV promoter
Observed lower expression levels and transfection efficiency when fused
How was the final pig transgene construct developed?
PSP promoter then replaced CMV promoter
Iserted into piggyBac transposon vector
How were the transgenic pigs developed?
1) Transgene vector and transposase expression vector were co- transfected into porcine fetal fibroblasts (PFFs) of male pigs
2) PCR used to detect for transgene
3) Four colonies with great EGFP expression used as donor cells for somatic cell nuclear transfer
4) Oocyte cell nucleus is removed
5) Fibroblast nucleus inserted into oocyte cell
6) Embryos are formed
What was determined from the transgenic pigs?
25/35 of the cloned piglets born were positive for
the transgene by PCR detection
5 had fragments of the ampicillin- resistance gene of the
transgene vector likely due to non specific integration
Efficiency of the transgene was very high: 80.6%
What did quantitative PCR show?
parotid gland > submandibular glands > sublingual glands for expression
