Assume that you are able to stably transform human cells with the vector. Insert

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?

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?

Created with SnapGene® (6057) NotI (6020) Spel PfIFI Tth111I (5) (6016) Bmtl 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

A. RNAi construct will be present in the hemizygous condition in the first generation after transformation. We can establish homozygous lines by PCR phenotypic analysis. However, there is no need for that in most of the cases. This is because RNAi constructs function in a dominant manner. i.e a single copy of the construct is sufficient to produce enough siRNA molecules to degrade that target transcript.

B. To confirm whether the RNAi construct is functional

i. We can check the transcript level of the target gene by qPCR

ii. We can check the transcript level of the target gene by Northern blotting

iii. We can check the protein level of the target gene by Western blotting

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