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Objectives for Gene Targeting in Canine HSCs

Objectives for Gene Targeting in Canine HSCs. Establish conditions for efficient targeting in canine D17 cells using eBFP/eGFP system Establish conditions for targeting CD34 + HSCs Establish a canine BM chimera with the EBFP target site Target endogenous loci in canine HSCs. I-SceI or Ani-I.

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Objectives for Gene Targeting in Canine HSCs

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  1. Objectives for Gene Targeting in Canine HSCs • Establish conditions for efficient targeting in canine D17 cells using eBFP/eGFP system • Establish conditions for targeting CD34+ HSCs • Establish a canine BM chimera with the EBFP target site • Target endogenous loci in canine HSCs

  2. I-SceI or Ani-I cis-acting region Pur eBFP I WPRE Pur CMV I WPRE CMV eBFP I-SceI or Ani-I intron eBFP-N eBFP-N or Ani-I Pur eGFP I WPRE CMV Targeting Strategy Foamy vector Integration Vector provirus selection of PurR cells Transduction with IDLV (I-SceI/Ani-I cleavage and eBFP targeting)

  3. Results Generation of FV-eBFP Control FV-eGFP FV-eBFP 293T transfected eBFP HT1080 transduced Estimated titer: 2X10e5 IU/ml (non-concentrated sup.) Estimated titer: ? D17 transduced 200l non-concentrated sup. MOI=1 200l non-concentrated sup. MOI=? eGFP

  4. eBFP expression in D17 transfected and puro-treated cells

  5. I-SceI or Ani-I cis-acting region SFFV-MGMT PGK eGFP SFFV-MGMT PGK eYFP or Ani-I Alternative ApproacheGFP-to-eYFP change and MGMT selection Foamy vector provirus Donor template vector (IDLV)

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