Breeding of Lanyu-derived miniature pigs harbouring the Ds-Red gene and its mtDNA analysis
Date Issued
2015
Date
2015
Author(s)
Chen, Sou-Fu
Abstract
DsRed, an Anthozoa-derived fluorescent protein with an excitation spectrum at 500 ~ 583 nm and a major peak at 558 nm has become a widely applied tool in the field of biomedical research. It has advantages such as faster maturation, less autofluorescence in animal tissues and better resistance to pH, making it ideal for multi-label colouring and dual-colour experiments with other fluorescent proteins. With the application of pronucleus injection, we were able to generate pigs harbouring the Ds-Red monomer protein. However, although the domestic pig (Sus scrofa domesticus or Sus domesticus) represents a great model animal, the size and weight of a regular pig (90 kg at 6 months), the expenses compared to smaller animals such as rodents and the difficulty of manipulation still pose a disadvantage. Hence, using smaller pigs such as the Lanyu-pig as an animal for human disease model has increased in numbers during the recent years. In most multicellular organisms, mtDNA is inherited from the mother (maternally inherited). Mechanisms for this include simple dilution, degradation of sperm mtDNA in the fertilized egg, and, at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism is, this single parent (uniparental) pattern of mtDNA inheritance is found in most animals. In recent years, mtDNA has been widely used for phylogenetic studies to estimate the genetic relationship among mammalian species. The objective of this study is to breed a new strain of miniature pigs harbouring the DsRed monomer protein and the analysis of the mitochondrial DNA. For this study, male European domesticated pigs expressing the DsRed protein and female Lanyu-derived strain were utilized to breed a cross-hybrid offspring of miniature pigs expressing the DsRed monomer protein. Fibroblast cells of each generation was cultured for further total DNA extraction and three sets of primers were designed for PCR analyses: DsRed (780 bp), for detection of the DsRed monomer, mtDNA Control Region (1380 bp), which is an area of non-coding DNA and is the most polymorphic region of the mtDNA genome, and mtDNA cytochrome B (1308 bp), which is commonly used to determine phylogenetic relationships within families and genera. PCR analysis using the DsRed primer show that some of the offspring from the four relating generations G1, G2, G3 and G4 harbouring the DsRed monomer protein gene, with an average germ-line transmission rate at 67%. The two pairs of mtDNA primers, which are Lanyu specific, showed that the progenies to be Lanyu derived, whether or not harbouring the DsRed monomer protein gene. Steady decrease of body weight at 20 weeks age from G1 to G3, with G2 showing a 76% decrease in body weight compared to G1, and G3 showing a 59% decrease in body weight compared to G2, imply the Ds-Red transgenic line was successfully miniaturized. Further compare the physiological parameter (body length, height, weight, at 8th and 20th week) between G3 and Lanyu pig indicates that G3 offspring fit the description of a “mini-pig”. Taken above, the data show successful breeding of Lanyu-derived pig expressing the DsRed monomer protein, indicating this new breed of DsRed-miniature pigs possess potentials for use as a new model animal in the field of biomedical studies and application.
Subjects
Ds-Red
miniaturization
Lanyu-derived pig
mitochondrial DNA (mtDNA)
SDGs
Type
thesis