Study On HAL Gene And OB Gene Of Tibet Mini-pigs, A New Laboratory Animal

In China, Tibet mini-pig, a kind of special plateau pig has great potential to becultivated into laboratory animal. HAL gene is also called RYR1 gene or CRC gene.There is close relationship between HAL gene and PSS (Porcine Stress Syndrome) orMH (Malignant Hyperthermia). HAL gene is the major gene that influences thesensitivity to stress. Leptin is one kind of hormone which is mainly secreted by whiteadipose tissue. The main function of leptin mainly secreted by white adipose tissue isto act on the bodyweight regulatory center of hypothalamus, and subsequently causeappetite decrease and weight losing. The abnormal expression of OB gene andresistance of leptin are closely relative with the morbidity of adiposity.Objective: The final goal of this project is to cultivate the Tibet mini-pig into anew laboratory animal. Thus, one objective of this study is to calculate the genotypefrequency of HAL gene, and then screen the pigs with HAL~NHAL~N genotype,HAL~NHAL~n genotype and HAL~nHAL~n genotype, gathering the basic data of Tibetminiature pig colony. In addition, to examine the feasibility of cultivating Tibetmini-pig into a kind of animal model for human diseases. Little bodily form andlowness of body fat are the most significant characteristics of Tibet mini-pig. Theseindicate that there should be difference on the mechanism of body weight controlbetween Tibet mini-pigs and other breeds of pigs. Firstly we want to find and optimize the methods to distill the total RNA from white adipose tissue of pig.Secondly compare the sequences of OB gene from several breeds of pigs by BLAST,getting the identity of Tibet mini-pig OB gene fragment. Finally we want to examinethe feasibility of establishing animal model of human adiposity and cardiopathy usingTibet mini-pigs.Methods: PCR-RFLP and RT-PCR were used in this study. Firstly bloodsamples were collected from 50 Tibet mini-pigs, followed by abstracting genomeDNA from the blood sample. HAL gene fragments amplified by PCR were digestedwith HhaⅠ.36 DNA samples of Duroc pigs and 20 DNA samples of Long-white pigswere done at the same time. The genotypes are divided into 3 kinds byelectrophoresis analysis, of which HAL~NHAL~N represented two bands (493bp and166bp), HAL~NHAL~n represented three bands (659bp, 493bp and 166bp), HAL~nHAL~nrepresented one band (659bp).White adipose tissue was collected from the body of Tibet mini-pig, and wasdived into fluid nitrogen immediately, and then stored at -70℃. cDNA of leptin genewere obtained by RT-PCR from the white adipose tissue total RNA, and then it wasused as a template. The leptin gene fragment was amplified by specific primer, andthen compared with human, mice and other pigs' leptin gene order after sequencing.Thus we can gather the information of the leptin gene order sequence signature ofTibet mini-pig.Conclusion: In all of 50 Tibet mini-pigs samples, none of HAL~n was found. TheHAL genotype frequency of Tibet mini-pig colony is 100% for HAL~NHAL~N, 0 forHAL~NHAL~n and HAL~nHAL~n. There is no HAL~n gene in the colony. Otherwise 4samples and 1 sample with HAL~NHAL~n genotype was detected from Duroc andLong-white pigs separately. There we make the conclusion that Tibet mini-pigs havehigher ability to defense the press. The results of OB gene sequencing of Tibet mini-pigs show that there is no mutant in the sequence of OB gene fragment. Theidentity between Tibet mini-pig and human, mouse, other breed of pig were 89%,83% and 99%, respectively. We find a single mutation in the sequence of Tibetmini-pig OB gene.