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| Construction of the Fosmid Genomic Library for the Lean Subline and the Fat Subline of Banna Miniature Inbred Pig (Sus scrofa) |
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Abstract Banna Miniature Inbred Pig (Sus scrofa) is the first large inbred mammal experimental animal. Its gene is highly homozygous, and genetic background is clear. And its gene has great potential in genetic breeding, functional gene research, human disease model and xenotransplantation. However, due to the high degree of inbreeding depression, the survival rate of offspring is low and genetic resources are very precious, and the preservation of the genetic information has scientific and practical significance. In this paper, the lean subline pig and the fat subline pig were taken as the research objects. The two subline pigs' DNA was extracted from agarose pre-embedding lumps, and sheared to the appropriate size (about 36~48 kb) by physical method. And then, the DNA fragments were used to construct the two sublines' Fosmid libraries by CopyControl? Fosmid Library Production kit. There were about 300 000 and 400 000 clones of the lean subline pig's and the fat subline pig's Fosmid library respectively, which covers the genome up to 4.4 and 5.9 fold. Basing on the upstream sequence of lean subline pig' heart-type fatty acid-binding protein gene, we designed the specific primers for grading PCR to screen the target clone in the library. Firstly, the first round PCR reaction using the original library bacteria solutions as template were conducted, which could screen the tube containing the target clones. When the tube which contained the positive target clones was confirmed, and then the positive tube bacterium were cultured. Then, the colonies of every plate were collected to one EP tube, and used as template to do PCR, to continue to screen the target tube. Like this, several rounds grading PCR were done until the target tube which contained 100~1 000 CFU per 10 μL LB medium was screened. Then this positive tube bacterium were cultured on LB medium plates. After 12-16 hours, the single colonies on LB medium plate were transferred to 96-well plate continued to culture as the same conditions as before. Then, the single colony bacteria solutions were used to do PCR to screen positive colony, and sequenced to confirm the single colony of bacteria containing target gene. Five single colonies that were identified by sequence analysis were abtained. Using the above method, we can get the fat subline pig's target single gene clone. We have successfully constructed the two subline pigs' Fosmid libraries, with a high rate of genome coverage, and the probability of screening to the target gene is 99% in theory. These Fosmid libraries can be used to preserve Banna Miniature Inbred Pig's genetic resources and carry out more deeply research on function and regulation of gene.
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Received: 14 June 2017
Published: 10 December 2017
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