Preparation Of A Transfected Goat Fetal Fibroblast Cell Stably Integrated Human Lactoferrin CDNA

In this study, 4.5 kb and 6.5 kb goatβ-casein gene 5′regulatory regions were cloned from goat genomic DNA by Long and Acute PCR, respectively. Following gel purification, the GBC6.5 and GBC4.5 PCR fragments were cloned into pMD18-T vector by T-A cloning and named pGBC4.5 and pGBC6.5, respectively, which are the same asβ-casein gene in GenBank (accession No. AF409096). Then we used the primers with restriction endonuclease recognition sites to amplify GBC6.5 and GBC4.5 again. The primers for GBC6.5 (6BSS, 6BSA) and GBC4.5 (4BSS, 4BSA) were contained Bsp EI recognition site (underlined) in 5′-terminal ends, and Sal I recognition site (underlined) in 3′-terminal ends to facilitate subcloning into MCS of the following plasmid vectors. Then GBC6.5 and GBC4.5 PCR products were digested with Bsp EI and Sal I and purified, and subsequently subcloned into the corresponding sites of the vector pEGFP-C1. The resulted plasmids were called p4.5EGFP and p6.5EGFP, respectively. Human lactoferrin(hLF) cDNA was amplified by PCR amplification from the plasmid pBL. In order to facilitate subcloning into MCS of the following plasmid vectors, primers used for hLF cDNA synthesis were designed to contain Sal I recognition site (underlined) in 5′-terminal ends and Bam HI recognition site in 3′-terminal ends (underlined). The PCR product of hLF cDNA was digested with Sal I and Bam HI overnight, then purified and cloned into the corresponding sites in the plasmid p6.5EGFP and p4.5EGFP. The resulted plasmids were designated as p6.5hLF-EGFP and p4.5hLF-EGFP and were sequenced completely by Invitrogen Inc.The expression vector p6.5EGFP and p4.5EGFP have the following advantages:①GBC4.5 (4.5 kb) consists of Promoter and enhancer regions; GBC6.5 is 6.5 kb 5′flanking sequence including exon 1, intron 1 and part of exon2.②The primers for GBC6.5 (6BSS, 6BSA) and GBC4.5 (4BSS, 4BSA) contained TAA stop codon (bold) in 5′-terminal ends to avoid forming fusion proteins with EGFP gene and protection base which are useful in digestion.③They contain reporter gene EGFP and selection gene-neo which are used to select positive cells.Then the caprine fetal fibroblast cells (FFC) were transfected with the vectors by lipofectamineTM 2000 and selected with G418 for 3~4 weeks. Furthermore, the G418 resistant cell lines, named p6.5hLF-EGFP-FFC and p4.5hLF-EGFP-FFC, were subsequently estimated by multiple PCR amplifications, and then the expression of reporter gene EGFP and chromosome karyotype were analyzed, The results indicated that the hLFcDNA were stably integrated into chromatin open regions and the karyotypes of both bp6.5hLF-EGFP-FFC and p4.5hLF-EGFP-FFC were normal. Therefore, it is suggested that the transgenic caprine fetal fibroblast cell lines, p6.5hLF-EGFP-FFC and p4.5hLF-EGFP-FFC, could be the reliable donor cells for the production of transgenic cloned goats which may secret human lactoferrin in the milk.