IRES mediated bicistronic vector expression in normal mouse epithelial mammary cell line

MUCCIN, KAISER G AND MUTTO A

Tahoe lake

Conferencia; Transgenic Animal Research Conference IX; 2013

UC Davis

β-casein gene has being widely used as a promoter to drive transgene expression in the mammary gland of transgenic domestic animals. In order to test vector functionality, an in vitro culture system of normal murine mammary gland cell line (NMuMG) was used. A 1960bp Human Lactoferrin cDNA (Gen Bank n: NM_002343) obtained from ATCC (NM_002343) encompassing the entire coding region was used. Gene was amplified from the original vector by PCR and cloned in vector pGEM-T easy (Promega, MO, USA). Primers contained in their extremes the recognition sequence for XhoI (New England Biolabs, cat r01465). Human lisozyme cDNA was obtained by RT-PCR using as a template mRNA of a in vitro macrophage culture obtained from peripheral blood. The 438 bp fragment corresponding to the complete codifying sequence of the gen was cloned in pGEM-T vector, but primers had different restriction sequences: forward MscI and reverse Eagl (NEB, cat: r05345 y r05055). The pIRES2-EGFP (Clontech Inc., USA) was used lika a backbone to construct the mammary gland expression vector. CMV promotor was replaced by the goat β-casein obtained from pBC-1 milk expression system (Invitrogen corp., CA, USA), obtained by PCR with Platinum TAQ DNApol (Gibco) and primer containing restriction sequences Asel (forward) and Nhel (reverse) (NEB, cat r05265 y r01315 respectively). Kozak sequence was added to each oligonucleotide used to amplified human genes in order to achieve better ribosomal reconnaissance and their correct expression. The presence and the correct orientation of the vector were verified by PCR and automatic sequencing respectively. Vector was spread and kept in E. coli Top Ten (Invitrogen, CA, USA). The mouse mammary epithelial cell line (NMuMG) used for gene transfection and expression studies was obtained from ATCC (CRL-1636). Cells at 70 % confluence were exposed to a mix of 5 μg DNA of bicistronic vector and 2μL Lipofectamin 2000 (Invitrogen, CA, USA) in 100μl OptiMEM (Gibco) without antibiotics for 4 hours at 37° C. Transfection media was removed and the culture plate wells (6 wells plate) covered with 50 mg/cm2 of Matrigel? Basement Membrane Matrix (Becton Dickinson, Franklin Lakes, NJ,USA). Lactogenic hormones (5μg.mL-1 Insulin, 10μg.mL-1 prolactin and 1μg.mL-1 Dexamethasone; Doppler et al. 1989) were added to culture media to induced vector expression. The fibroblast colonies resistant to neomycin were isolated and grown in Dulbecco`s modified Eagle`s medium (DMEM) with 4.5g.L-1glucose (Gibco BRL, Rockville, UT, USA), 10% heat inactivated fetal calf serum (HyClone, UT, USA) and 50μg.mL-1 gentamycin (Gibco BRL) and 25ug/ml Neomycin (Gibco BRL). The presence of human proteins (lactoferrin and lisozyme) was determined in samples obtained from culture media of induced transfected NMuMG cells. Culture media (100 microl) was mixed with cracking buffer. The samples were resolved in SDS-PAGE and the proteins transferred to a Hybond C blotting membrane (Amersham, USA). To detect the recombinant proteins and controls, the membrane was blocked overnight in TBS buffer (50mM Tris-HCl, pH 7,6; 150mM NaCl) with 5% milk. The primary antibodies were either rabbit anti human lactoferrin or lisozyme (Dako, Cytomation, USA) diluted 1/3000 in TBS solution with 5% milk. The membrane was washed three times with TBS Tween 20 (0,05 Tween 20 in TBS). The second antibody was a Goat anti rabbit IgG Horseradish Peroxidase conjugate (Gibco, CA, USA) and developed with Supersignal West Pico Chemiluminiscent (Pierce, USA). The reaction was revealed using a X ray film (Kodak, Japón), according to the manufacturer´s instruction