Gene Targeting Toward β-lactoglobulin Gene In Goat

Production of transgenic domestic animals which can be used to produce pharmaceuticalproteins or humanized milk is the popular research topics in recent years. Target genes can beknockout to disrupt its expression and the interested gene can be inserted into the specificlocus of the genome and expressed under the control of the internal regulatory elements withgene targeting techniques. As the main whey proteins in human milk, α-lactalbumin (ALA)was thought as the rich sources of essential amino acids, such as tryptophan, lysine andcysteine, especially for infants. It has been found that ALA may take part in the prevention ofgastrointestinal infections among neonates. In addition, as the main whey protein of theruminant milk, BLG was not contained in the milk of the human, and was considered as themain allergen of the cattle or goat milk. With the aim of producing transgenic goat which cansecrete humanized milk, we want to knockout the BLG gene in goat and/or to replace it withhALA using the gene targeting combined with somatic cell nuclear transfer techniques.1. Firstly, we cloned the human ALA gene by PCR with serum genomic DNA as template.Meanwhile, in order to clone the cDNA of hALA, we integrated the hALA into the eukaryoticexpression vector pIRES2-EGFP. Then, the vector was transfected into the293T cells toconstruct the surrogate cells from which the total RNAs were extracted to clone the cDNA byRT-PCR. This protocol can be served as an alternative method to clone cDNA which onlyexpressed in the tissues not so conveniently to collect.2. Three fragments of BLG in goat were cloned by PCR, and inserted into the fluorescentreporter vector pAdTrack-RFP. After recombination with pAdEasy-1in BJ5183, theadenoviral vector pAd-B51RFP, pAd-B52RFP and pAd-B53RFP were constructed in whichthe expression of mRFP was directed by the regulatory element, respectively. Then theadenovirus were packaged and used to infect the goat mammary epithelial cells. Wheninduced with corresponding hormones the expression of mRFP can be dtectected in the goatmammary epithelial cells, which showed that the BLG fragments were cloned successfully.3. Four targeting vectors pBAT, pBATGFP, pBATM and pBAcT were constructedsuccessfully identified by enzymic digestion and sequencing. In order to assay the expressional elements in pBATM, the hALA combined with5’ and3’ flanking regions of BLGin pBATM was sub-cloned to replace the RFPPA in pAdTrack-RFP to construct theadenovirus. Induction the lactation of goat mammary epithelial cells infected with theadenovirus. The hALA was detected by western bloting in the culture medium, which furtherdemonstrated that the pBATM were constructed correctly.4. Transfecting the goat fibroblast cells with the linearized four targeting vector andknockout vector pBLG2T, respectively. After selection with G418combined with ganciclovir(GCV),2(B412F1and B7732F6） and3(T229F2, T1041F1and T7963F6） strains of usabletargeted cells with normal karyotype were got in the group transfected with pBAT or pBLG2T,respectively. Unfortunately, GFP almostly cannot be detected in the clones transfected withpBATGFP, which suggested that it cannot be used to reject the contaminant wild cells mixedin the selected positive cell strains.5. With the cells from the five selected targeted strains as donors, the recombinantembryos were transferred into the oviducts of the recipient goats after cultured24h in vitro.Totally about18goats were transferred and8pregnant goats were detected about45dayslater and3full term development fetuses were got finally. After detection with PCR andsouthern blot, one fetus was BLG knockout and one was hALA targeted integrated.