Assume that you are able to stably transform human cells with the vector. Insert
ID: 256999 • Letter: A
Question
Assume that you are able to stably transform human cells with the vector. Insertion may occur more than one time, but only happens once at each locus. Transformed cells are thus hemizygous (heterozygous) for the insert.
a. Would you expect the RNAi to be a dominant or recessive in terms of phenotype. That is, will it work in the hemizygous state or does it have to be homozygous? (1pt)
b. Give two methods you could use to determine if the RNAi construct was affecting the expression of native (produced by the cell) XM_011528386.1? (2pts)
Created with SnapGene® (6057) NotI (6020) Spel PfIFI Tth111I (5) (6016) Bmti PvuI (136) (6012) Nhel (6008) SphI Fsp (155) BsaAI - DraIII (412) (6002) PstI Sbfl (5977) Ahdl BtgZI (413) (5902) Apal (5898) PspoMI (5847) BstEII (5753) BssHII 6000 (5749) Mrel-SgrAI (5678) PasI (5623) Agel ocS terminator (5301) BsmBI (5279) Xbal (5273) BamHI (5267) HindIII (5262) BspDI Clal XcmI (1239) (5151) Pacl pKANNIBAL 6063 bp (4466) KpnI (4462) Acc65I (4456) EcORI 4450) PaeR7I- PspXI TIiI Xhol BtgI (1635) 4326) BmgBI EcoRV (1767) (4263) PshAI 4053) AccI (4027) HincII (3935) BsaBI (3856) StyI 3000 (3816) Scal (3682) Bsal (3664) Stul (3332) Mlul (3097) Not (3089) Sac (3087) Eco53kI BspQI SapI (2806) (3058) Sfil MscI (3052)Explanation / Answer
1. RNAi can work in hemizygous state as it requires mRNA to be produced. If mRNA is produced in a hemizygous state from one of the alleles and that allele uses some significant effect then RNAi would certainly work.
2. There is a PDK intron in the vector. Thus the native mRNA won't have any such tag. Hence, a Northern blotting of the transcriptome will reveal whether the RNA is still present or it has been degraded because of RNAi. Concurrently reverse transcriptase PCR can be done to cross check the same.