Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line.
A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.
Tan
W
W
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Guoxuexiang 37, Chengdu 610041, P.R. China.
Chen
Y
Y
Zhang
L
L
Lu
Y
Y
Li
S
S
Zeng
R
R
Zeng
Y
Y
Li
Y
Y
Cheng
J
J
D000818:Animals; D002681:China; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D008099:Liver / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013552:Swine; D013556:Swine, Miniature
D018076:DNA, Complementary; D012313:RNA