Analysis Of LAMININ Expression Pattern In PiPSCs And Molecular Cloning Of An Alternative Splicing

Laminin(LN)is an important component of cell membrane,which was used as cell matrix to maintain self-renewal and pluripotency of pluripotent stem cells.LN family has the15 different subtypes,each subtype consists of three chains(?,?,?)that form a Laminin heterotrimer.Laminin A5(LAMA5)is one of the LN family and plays an important role in maintaining the growth of pluripotent stem cells.The aim of this study was to detect the expression profile of LAMININ in different porcine tissues and cells;to determine the LAMININ expression during stem cell differention;to confirm the presence of pig LAMA5 gene and the expression change through bioinformatics analysis and molecular cloning;to discover and molecular clone porcine Laminin B1–a(LAMB1-a)alternative splicing isoform.This study enriches the information of culture conditions for porcine pluripotent stem cells,and provides the theoretical basis for porcine cell reprogramming.The main results obtained in this study are as follows:1.Expression profile of LAMININ in porcine tissues and cellsWe detect the expression profile of LAMININ in different porcine tissues(including liver,heart,lung,stomach,brain,testis,fat,and granulosa-cell),cells(hepatocytes,adipocytes,porcine fibroblasts)and pluripotent stem cells(DOX-iPS,PS11,PS23)by RT-PCR.Results showed that there were significantly differences between the expression of LAMININ in tissues and cells,By comparing the tissue source,LAMININ expression specificity in cells was more prominent,in which the expression of LAMININ in PEF cells was characterized as follows: LAMA4/5-LAMB1/2-LAMC1/2(LN-411,LN-421,LN-511,LN-521);The expression of LAMININ in pig iPS cells was characterized as: LAMA1/5-LAMB1-a / 2-LAMC1(.LN-11a1,LN-121,LN-51 a1,LN-521).2.Biological analysis and cloning of porcine LAMA5There is no sequence information and reports on porcine LAMA5 gene.Therefore,we used NCBI and UCSC databases to analyze the LAMA5 gene sequence of human,cattle and goats,and to predict the possible sequence of porcine LAMA5 and to locate it in pig genome database(Sscrofa11.1)within the RPS21 and CABLES2 genes.The sequence of porcineLAMA5 was confirmed by DNAMAN(V6)software,and designed the specific primers.The porcine LAMA5 gene was cloned from porcine lung tissue cDNA with length of 731 bp.The sequencing result was consistent with the human and bovine LAMA5 sequences,and the sequence was uploaded to GENBANK for accession number: KY622023.3.The temporal and spatial expression of LAMININ during pig iPS reprogrammingThrough RT-PCR test the expression of LAMININ,found that LAMB1 appeared two specific bands only in iPS cells.In order to further study the properties of LAMB1,through cloning the two specific bands(LAMB1 505 bp,LAMB1-a 639 bp).The results showed that LAMB1-a was a new variable spliceosome with the LAMB1-X1 and LAMB1-X2 sequences in NCBI.In order to further investigate the correlation between LAMB1 and pluripotent stem cells,selected the PEF and pig pluripotent cells DOX-iPS samples in different period of differentiation.The results showed that the expression of LAMB1-a gradually decreased with the morphological changes of the pluripotent cells.After the re-use of DOX-iPS medium,the expression of LAMB1-a gradually decreased restore when the morphology recovered again.In the detection,it was also found that the expression of LAMA4 was significantly increased in the process of iPS reprogramming,and the expression of LAMA1 was gradually decreased in the process of cell differentiation.In conclusion,the expression of LAMININ in pig pluripotent stem cells was identified,namely,LN-11a1 / LN-121 and LN-51a1 / LN-521.The presence of pig LAMA5 gene was confirmed,analyzed and cloned.Found LAMB1-a variable spliceosome,and verified the corresponding relations between genes and induced pluripotent stem cells.