Knock-out, Knock-in, Knock-down Dr. Thorsten Buch by 0G37k53J

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									          Knock-out, Knock-in, Knock-
                     down
          Dr. Thorsten Buch, Neuroimmunology
              Thorsten.Buch@neuroimm.unizh.ch
                          635 3709




Bio 426
              Knock-Outs: Problem 1

          Embryonic Lethality

          breeding heterozygous animals does not yield homozygous mutants

          offspring ratio: heterzygous : WT       2:1


          options:

          start a project on embryonic development

          in immunology:
          -make blastocyst complementation
          -adoptive transfer of fetal liver cells into irradiated hosts




Bio 426
                  How to overcome it!

          The development of conditional mutagenesis

          cell type specific mutagenesis

          inducible mutagenesis

          cell type specific inducible mutagenesis




Bio 426
                       Conditional Targeting I
                                Cre recombinase

              loxP site 5'-ATAACTTCGTATA ATGTATGC TATACGAAGTTAT-3'
              Cre recombinase: recombinating enzyme of the phage P1




          3        4        5                   3   4       5

                        Cre mediated deletion           Cre mediated inversion

                                                    4
               3        5                       3           5




      other recombination system: Flp recombinase, frt sites
Bio 426
               Conditional Targeting II
                  celltype specific mutagenesis

                                                  Cre

                                                        Cre




          loxP flanked gene                  tissue specific Cre




                                 Cre

                                       Cre




                     tissue-specific gene deletion
Bio 426
                       Conditional Targeting III
                                      targeting strategy
                              E             E

                                      3     NeoR          4           5    HSV-TK
              E                                                              E               frt site

                       1          2         3         4       5           6                  loxP site
              5‘ probe
                                          12 kb                           3‘ probe



                                      E           E                                      E
          E
                   1          2             3      NeoR           4           5      6

                       5 kb                                                   7 kb


          place a cutting site next to the single loxP
Bio 426
               Conditional Targeting IV
                             targeting strategy
                             E       E                               E
      E
                                 3       NeoR
                                                                         frt site
               5 kb                                       7 kb
                                          Flp
                                          recombination                  loxP site
          E                      E                               E
                                     3

                  5 kb                            6 kb
                                          Cre
                                          recombination
          E                      E                        E



                      5 kb                      4 kb

Bio 426
              plan your Southern strategy all the way through
                 Targeting: Step 0


          •go to ENSEMBL.org, look up BAC of your locus

          •order BAC (CHORI.org etc.)

          •immediately make glycerol stock of BAC bacteria




Bio 426
                     Targeting: Step 1
                     Which exon to „flox“?
          promoter



                      1        2    3     4     5        6


              ligand binding   enzymatic transmembrane
                                 center       region




          -taking out the transmembrane region
           can lead to soluble receptor
          -placing loxP in promoter is dangerous
Bio 426
                        Targeting: Step 1
                          Which exon to „flox“?
                   • check splicing carefully by hand
                   • do not trust intron-exon structure of databases
                   • check also for natural splice variants

                                       intron

                       2                                          1
 ttt tca tta ttc ctt agg tragttgtggttctrayytacagaaaagccagyyyyyncagg gta ttc agg ctg gat

                              0   0    2   1       2   1 0 0 1   2
                          1        2           3       4   5     6


                                           Bad


Bio 426
            Targeting: Step 1
              Which exon to „flox“?
          •check splicing carefully
          •check also for natural splice variants


                  0   0    2   1       2   1 0 0 1   2
              1        2           3       4   5     6



                  not perfect but better...




Bio 426
                              Targeting: Step 2
                       design and test Southern blot
                               E            E

                                      3     NeoR          4           5    HSV-TK
              E                                                              E               frt site

                       1          2         3         4       5           6                  loxP site
              5‘ probe
                                          12 kb                           3‘ probe



                                      E           E                                      E
          E
                   1          2             3      NeoR           4           5      6

                       5 kb                                                   7 kb

                  • amplify probes, make test Southern
                  • repeat if necessary by moving the probe
Bio 426
                  • do not clone vector before Southern works!!!
                        Targeting: Step 3
                 design and check arms in silico
                                     floxed
                           5´arm     exon                  3´arm


                                      3       NeoR     4      5    HSV-TK



                  2-4 kb           0.5 – 2 kb           4-6 kb


                                                     avoid repeats in the arms!!!

                                                      do not destroy splice
                                                      branch site in the
                                                      introns around the
                                                      floxed exon

   ttt tca tta ttc ctt agg tragttgtggttctrayytacagaaaagccagyyyyynca ggg gta ttc agg ctg gat
Bio 426
          Cloning Targeting Vectors
                                                          clone last
                                     XhoI   SalI
                                                          •amplify arms from BAC
                       3               4    5              (use high fidelity enzyme)

                                                          •check that order of cloning
                                                           works

                                                          •clone into vector

          XhoI SalI          BamHI                        •sequence exons

                      NeoR      HSV-TK      pRapidflirt   •test integrity of vector with
                                                           7-10 restriction enzymes




                                Go ahead!!!
Bio 426
          Targeting vectors from BACs I

 What are BACs
         Vectors used to clone DNA fragments (100- to 300-kb insert size;
         average, 150 kb) in E. coli cells
         Based on naturally occurring F-factor plasmid found in the bacterium E. coli

 Why transgenes/targetings with BACs?

           BACs can be ordered easily from semi-public sources, result of
           sequencing of mouse genome
           BACs are easily modified by homologous recombination (E/T cloning)
           large homology regions


Attention: difficult to impossible to design Southern strategy
                      -> make BAC smaller

Bio 426
                 Problems of Knock-outs

          Embryonic Lethality

          knock-out of such a gene does not result in viable homozygous mice
          such genes are identified by breeding:
             -ratio of genotypes in offspring is: heterozygotes : WT 2:1




Bio 426
                       Conditional Targeting I
                                Cre recombinase

              loxP site 5'-ATAACTTCGTATA ATGTATGC TATACGAAGTTAT-3'
              Cre recombinase: recombinating enzyme of the phage P1




          3        4        5                   3   4       5

                        Cre mediated deletion           Cre mediated inversion

                                                    4
               3        5                       3           5




      other recombination system: Flp recombinase, frt sites
Bio 426
               Conditional Targeting II
                  celltype specific mutagenesis

                                                  Cre

                                                        Cre




          loxP flanked gene                  tissue specific Cre




                                 Cre

                                       Cre




                     tissue-specific gene deletion
Bio 426
                       Conditional Targeting III
                                      targeting strategy
                              E             E

                                      3     NeoR          4           5    HSV-TK
              E                                                              E               frt site

                       1          2         3         4       5           6                  loxP site
              5‘ probe
                                          12 kb                           3‘ probe



                                      E           E                                      E
          E
                   1          2             3      NeoR           4           5      6

                       5 kb                                                   7 kb


          place a cutting site next to the single loxP
Bio 426
               Conditional Targeting IV
                             targeting strategy
                             E       E                               E
      E
                                 3       NeoR
                                                                         frt site
               5 kb                                       7 kb
                                          Flp
                                          recombination                  loxP site
          E                      E                               E
                                     3

                  5 kb                            6 kb
                                          Cre
                                          recombination
          E                      E                        E



                      5 kb                      4 kb

Bio 426
              plan your Southern strategy all the way through
                 Targeting: Step 0


          •go to ENSEMBL.org, look up BAC of your locus

          •order BAC (CHORI.org etc.)

          •immediately make glycerol stock of BAC bacteria




Bio 426
                     Targeting: Step 1
                     Which exon to „flox“?
          promoter



                      1        2    3     4     5        6


              ligand binding   enzymatic transmembrane
                                 center       region




          -taking out the transmembrane region
           can lead to soluble receptor
          -placing loxP in promoter is dangerous
Bio 426
                        Targeting: Step 1
                          Which exon to „flox“?
                   • check splicing carefully by hand
                   • do not trust intron-exon structure of databases
                   • check also for natural splice variants

                                       intron

                       2                                          1
 ttt tca tta ttc ctt agg tragttgtggttctrayytacagaaaagccagyyyyyncagg gta ttc agg ctg gat

                              0   0    2   1       2   1 0 0 1   2
                          1        2           3       4   5     6


                                           Bad


Bio 426
            Targeting: Step 1
              Which exon to „flox“?
          •check splicing carefully
          •check also for natural splice variants


                  0   0    2   1       2   1 0 0 1   2
              1        2           3       4   5     6



                  not perfect but better...




Bio 426
                              Targeting: Step 2
                       design and test Southern blot
                               E            E

                                      3     NeoR          4           5    HSV-TK
              E                                                              E               frt site

                       1          2         3         4       5           6                  loxP site
              5‘ probe
                                          12 kb                           3‘ probe



                                      E           E                                      E
          E
                   1          2             3      NeoR           4           5      6

                       5 kb                                                   7 kb

                  • amplify probes, make test Southern
                  • repeat if necessary by moving the probe
Bio 426
                  • do not clone vector before Southern works!!!
                        Targeting: Step 3
                 design and check arms in silico
                                     floxed
                           5´arm     exon                  3´arm


                                      3       NeoR     4      5    HSV-TK



                  2-4 kb           0.5 – 2 kb           4-6 kb


                                                     avoid repeats in the arms!!!

                                                      do not destroy splice
                                                      branch site in the
                                                      introns around the
                                                      floxed exon

   ttt tca tta ttc ctt agg tragttgtggttctrayytacagaaaagccagyyyyynca ggg gta ttc agg ctg gat
Bio 426
          Cloning Targeting Vectors
                                                          clone last
                                     XhoI   SalI
                                                          •amplify arms from BAC
                       3               4    5              (use high fidelity enzyme)

                                                          •check that order of cloning
                                                           works

                                                          •clone into vector

          XhoI SalI          BamHI                        •sequence exons

                      NeoR      HSV-TK      pRapidflirt   •test integrity of vector with
                                                           7-10 restriction enzymes




                                Go ahead!!!
Bio 426
                Program
          • Knock-out
            –   what to knock out
            –   alternative splicing
            –   lineage ablation through knock-out
            –   NeoR effects
          • The Paper
          • Knock-in
            – dirty knock-in
            – Cre-loxP
            – removal of NeoR
          • Knock-down
            – haploinsufficiency
            – siRNA



Bio 426
                      Knock-Outs

          Main aim:

          -abrogating the function of a given gene or genetic element

          replacement of whole gene by NeoR
              often not possible because of size

          replacement of part of a gene by NeoR
                  Which part?

          replacement of promoter, silencer, enhancer, or locus
          control elements by NeoR

          inactivating a developmental pathway = lineage ablation




Bio 426
          Targeting: Which part to „knock-out?“


                                                                  signal
            promoter                                           transduction


                        1        2    3     4     5        6


                ligand binding   enzymatic transmembrane
                                   center       region




Bio 426
          Targeting Design: Splicing Problems

              Alternative Splicing can result in residual protein
              expression with unexpected results

              -no phenotype

              -mild phenotype (knock-down)

              -constitutive active protein

              -dominant negative protein




Bio 426
          Targeting Design: Splicing Problems

                   • check splicing carefully by hand
                   • do not trust intron-exon structure of databases
                   • check also for natural splice variants

                                       intron

                       2                                          1
 ttt tca tta ttc ctt agg tragttgtggttctrayytacagaaaagccagyyyyyncagg gta ttc agg ctg gat

                              0   0    2   1       2   1 0 0 1   2
                          1        2           3       4   5     6


                                           Bad


Bio 426
          Targeting Design: Splicing Problems II


                       0   0    2   1       2   1 0 0 1   2
                   1        2           3       4   5     6




                       not perfect but better...




Bio 426
          Lineage ablation through Knock-Outs

               Many genes are crucial for the development of a
               particular cell lineage

               knock-out of such a gene results in absence of the
               respective lineage from the mouse




Bio 426
              Lineage ablation: preTCR deficiency
                    CD4-
                    CD8-


                                       pre T cell
                    CD4+               receptor     pre T cell receptor
                    CD8+
                    TCRb+                           necessary for T cell
                                                    development

                    CD4+                            knock-out of pTa leads to
                    CD8+
                    abTCR                           absence of late thymocytes and
                    +
                                                    peripheral T cells


               positive/negative
                  selection




                                   CD4+
          CD8+
                                   abTCR
          abTCR                    +
          +




Bio 426
     A CFTR Mutation for Cystic Fibrosis




           50% lower expression level
           no conclusion can be drawn
Bio 426
          The CD3 epsilon knockout-
                 or how not to do it!




                                almost no T cells in periphery!




Bio 426
          The CD3 epsilon II


               knocked-out


               what about this?

               what about this?




Bio 426
          The CD3 epsilon knockout III




            CD3delta and gamma are close to CD3 epsilon
            What about Eva1?


Bio 426
                       The Cre/loxP System

              loxP site 5'-ATAACTTCGTATA ATGTATGC TATACGAAGTTAT-3'
              Cre recombinase: recombinating enzyme of the phage P1




          3        4       5                   3    4       5

                       Cre mediated deletion            Cre mediated inversion

                                                    4
               3       5                       3            5




      other recombination system: Flp recombinase, frt sites
Bio 426
          Use of Cre: Remove NeoR


                 1      2      NeoR          4        5   6

                                      Cre mediated
                                      recombination




                 1      2      4      5      6



          The NeoR cannot influence the experiment anymore




Bio 426
            Knock-in I: targeted expression

                                                            Targeting
                             GFP    NeoR       3       4
                                                            Vector
                                               Cross-over
          genomic
          locus                1   2       3       4


                                       Homologous Recombination (HR)



          insertion
                      Prom   GFP   NeoR    3       4
          transgene




Bio 426
          Knock-in II: point mutation
                        AT


                 1       2      NeoR          4        5     6

                                       Cre mediated
                                       recombination
                        AT


                 1       2      4      5      6



          Point mutations simulating mutations from human
          patients can now be simulated. Only loxP remains



Bio 426
              Knock-in III: purposes

          point mutations
             -imitiation of human disease
             -mutating tyrosines
             -mutating enzymatic centers

          reporter genes: lacZ, GFP, YFP, RFP, dsRED

          ablation genes: diphtheriatoxin A, thymidinkinase

          Cre recombinase




Bio 426
          Knock-down: Haploinsufficiency

           The definition
           -One functional copy of a given gene is not enough to maintain a
           protein level which is sufficient for the complete function
           -dominant phenotype by inheritance of one „knock-out“ allele

           In disease
           Cleidocranial dysostosis (CBFA1)
           Huntington's disease (HD)
           Polydactyly
           Marfan syndrome (FBN1)




Bio 426
          Haploinsufficiency: Example
                              +/-




Bio 426
                     Knock-down: siRNA
   Dicer is an RNAse that cleaves dsRNA into
   short double-stranded RNA fragments
   called small interfering RNA (siRNA) about
   20-25 nucleotides long, usually with a two-
   base overhang on the 3' end.




   RISC is the complex which mediates RNA
   interference by degrading the target DNA.
   It´s catalytically active enzyme is called
   argonaute




Bio 426
                    siRNA/knock-down

                              The System
          H1 promoter
                              -ca. 20-25 bp RNA hybridizing to
                              target can trigger degradation of
                              target

                              -this RNA can be produced as
                              hairpins from transgenes

                              -you need polymerase III promoters
                              (H1/U6) which have exact start and
                              stop sequences




Bio 426
           siRNA/knock-down
          in vitro very efficient!
          one needs to find the right sequence  algorithms

             GOI (after 3 failed trials (8 sequences))
                                                               IL-23R knockdown on mRNA level
                                                       40000
                                                                                  untransfected
                 IL-23R expression level, normalized
                                                                                  IL-23R/pElacZ
                                                                                  IL-23R/pEIL-23R 8-1
                                                       30000
                                                                                  IL-23R/pEIL-23R 10-1
                                                                                  IL-23R/pEIL-23R 11-1

                                                       20000




                                                       10000
                                                                                  Nearly 95% silencing


                                                          0
                                                                                    Z
                                                                                    d




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                                                                                  -1



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                                                                                 8-
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                                                                                 te




                                                                               10



                                                                               11
                                                                              ec



                                                                             El



                                                                              R



                                                                            R



                                                                            R
                                                                          23
                                                                           sf



                                                                           /p




                                                                         23



                                                                         23
                                                                        an



                                                                       3R



                                                                        L-



                                                                      L-



                                                                      L-
                                                                     EI
                                                                     tr



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                                                                   EI



                                                                   EI
                                                                 un




                                                                  /p
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                                                                /p



                                                                /p
                                                              3R



                                                             3R



                                                             3R
                                                           -2



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                                                          -2
                                                        IL



                                                       IL



                                                       IL




          in vivo data conflicting!
          does not work efficiently in lymphocytes
Bio 426
                Summary
          • Knock-out
            –   what to knock out
            –   alternative splicing
            –   lineage ablation through knock-out
            –   NeoR effects
          • The Paper
          • Knock-in
            – dirty knock-in
            – Cre-loxP
            – removal of NeoR
          • Knock-down
            – haploinsufficiency
            – siRNA



Bio 426

								
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