Construction of pBAC-hLF-hLZ-Neo expression vector: The procedure of modifying t

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Construction of pBAC-hLF-hLZ-Neo expression vector: The procedure of modifying the pBAC-hLF-hLZ-Neo construct was divided into three distinct steps, which were each verified by PCR and sequencing with primers. The first step is a replacement of hLF coding sequence with the one for hLZ, thus the targeting construct pMD19-hLZ-Zeo was linearized and electroporated into competent SW102 cells containing hLF BAC for recombination. Ten zeocin resistant clones were picked and verified by PCR with hybrid primers P1, which bind to the hLF 50 408 Transgenic Res (2012) 21:407–414 123 flanking regions and hLZ genomic sequence, respectively. All ten clones produced a band of the expected length (703 bp). No product was amplified from unmodified hLF BAC. The PCR-positive SW102 bacterial cells were made competent for the next procedure. In the second step, the neomycin resistance cassette, which allows stably transfected eukaryotic cells to be selected using G418 and expresses kanamycin resistance in E. coli, was inserted into the hLF BAC as a selectable marker for future SCNT. Recombineering was performed and verified as described above. The amplified products were 2,541 bp for BAC clones that recombination occurred at the correct location, and 1,960 bp for the negative control. In the final step, the zeocin cassette flanked by two direct repeat FRT sites was removed after transient expression of FLP recombinase in E. coli strain SW102. PCR analysis was performed across the recombination site, and the amplified products of positive clones were found to be 1,129 bp, about 1 kb smaller than the negative control because of the deletion of a zeocin cassette. P4 primers were prepared for digoxigenin (DIG)-labeled probes for Southern blot analysis . The final modified pBAC-hLF-hLZ-Neo construct contained a 90-kb 50 flanking region of the hLF gene, the 4.8-kb hLZ genomic fragment with a single FRT site retained, a 30-kb 30 flanking region of the hLF gene, and a Neo cassette for future SCNT.

What is the purpose of inserting neomycin resistance gene? What is the purpose for zeocin as it pertains to this experiment? explain the purposes that they serve to advance the experiments- when are they used for selection or to recognize what outcome?)

for more info read full article "High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC" Shen Liu

Explanation / Answer

The purpose of inserting neomycin resistance gene is to: select the stably transfected eukaryotic cells to be selected using G418 and expresses kanamycin resistance in E. coli.

The zeocin cassette flanked by two direct repeat FRT sites was removed after transient expression of FLP recombinase in E. coli strain SW102, as the the FLP protein recombined FRT sites previously integrated into the lead to excision of a selectable marker, the neo gene, (Lyznik, 1996)

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