1 Introduction ................................................ 1
1.1 Overview of a gene targeting project .................. 1
1.2 Genome engineering via homologous recombination ....... 2
1.3 The design of targeting constructs .................... 3
1.3.1 Positive and negative selection strategies .... 4
1.3.2 Counter selection strategies .................. 4
1.3.3 Promoterless cassettes to enrich for
targeted clones ............................... 5
1.3.4 Polycistronic targeting vectors ............... 5
1.4 Allele Design ......................................... 6
1.4.1 Expression alleles ............................ 7
1.4.2 Constitutive knockout alleles ................. 7
1.4.3 Site-specific recombination and the FLEx
switch ........................................ 9
1.4.4 Conditional knockout alleles ................. 10
1.5 The generation of targeting constructs by
recombineering ....................................... 13
1.6 Screening strategies ................................. 14
2 Material and methods ....................................... 15
2.1 Technical equipment .................................. 15
2.2 Consumable materials ................................. 16
2.3 Chemicals, Kits and Enzymes .......................... 16
2.4 Antibiotics .......................................... 17
2.5 Radioactive material ................................. 18
2.6 Solutions, Buffers and Media ......................... 18
2.7 Antibodies ........................................... 21
2.8 Synthetic oligonucleotides ........................... 22
2.9 Plasmids ............................................. 25
2.10 Cell lines ........................................... 25
2.11 Bacterial strains .................................... 26
2.12 Methods .............................................. 27
2.12.1 Cultivation of bacteria ...................... 27
2.12.2 Preparation and transformation of
electrocompetent cells ....................... 27
2.12.3 Retransformation of plasmid DNA .............. 27
2.12.4 Small-scale preparation of plasmid DNA ....... 28
2.12.5 Large-scale preparation of plasmid DNA ....... 28
2.12.6 Restriction digest ........................... 28
2.12.7 Ethanol precipitation ........................ 28
2.12.8 Standard, proofreading and long range PCR
(LRPCR) amplification ........................ 29
2.12.9 Colony PCR screening ......................... 29
2.12.10 Purification of PCR products ................. 29
2.12.11 Phenol-chloroform extraction ................. 30
2.12.12 Dephosphorylation and Ligation ............... 30
2.12.13 Agarose gel electrophoresis .................. 30
2.12.14 Gel purification of DNA fragments from
agarose gels ................................. 30
2.12.15 DNA sequencing ............................... 30
2.12.16 Extraction of RNA from ES cells .............. 31
2.12.17 cDNA Synthesis and RTPCR ..................... 31
2.12.18 DNA extraction from ES cells ................. 31
2.12.19 Southern blotting ............................ 32
2.12.20 Generation of radiolabeled DNA probes ........ 32
2.12.21 Southern hybridization of radiolabeled DNA
probes ....................................... 32
2.12.22 Recombineering proficient bacterial cells
for electroporation .......................... 33
2.12.23 Cultivation of ES cells ...................... 33
2.12.24 Freezing of ES cells ......................... 34
2.12.25 Electroporation of ES cells .................. 34
2.12.26 Hprt selection assay ......................... 34
2.12.27 ES cell colony assay ......................... 34
2.12.28 Protein extraction from ES cells (Mini
Nuclear Extracts) ............................ 35
2.12.29 Separation of proteins on polyacrylamide
gels ......................................... 35
2.12.30 Western blot analysis ........................ 36
3 Hprt targeting with asymmetrical targeting constructs ...... 37
3.1 Introduction ......................................... 37
3.2 Results .............................................. 40
3.2.1 Cloning of the constructs for the targeting
of the Hprt gene ............................. 40
3.2.2 Generation of LongRange PCR amplified
targeting constructs ......................... 44
3.2.3 Targeting of the Hprt gene in Rl ES cells .... 47
3.3 Discussion ........................................... 55
3.3.1 Subcloning by recombineering of two linear
PCR fragments ................................ 55
3.3.2 Generation of targeting constructs
containing PCR amplified homology arms ....... 56
3.3.3 Generation of PCR amplified targeting
constructs ................................... 57
3.3.4 Knockout of Hprt in Rl ES cells with LRPCR
amplified targeting constructs ............... 57
3.3.5 The methylation of targeting construct DNA ... 58
3.3.6 The effect of a counter selection cassette ... 59
3.3.7 Counter selection is dispensable for
promoterless targeting construct ............. 61
3.3.8 Summary and outlook .......................... 61
4 Novel conditional knockout cassettes ....................... 63
4.1 Introduction ......................................... 63
4.1.1 The FLEX cassette ............................ 64
4.1.2 The TIM cassette ............................. 65
4.2 Results: FLEX ........................................ 66
4.2.1 Cloning of a Phf8 - FLEX targeting
construct .................................... 66
4.2.2 Targeting of the Phf8 gene with the FLEX
cassette ..................................... 68
4.2.3 Analysis of the Phf8 - FLEX allele after
Cre-mediated inversion ....................... 68
4.3 Discussion: FLEX ..................................... 72
4.3.1 The FLEX cassette is a "conditional-first"
kockdown cassette ............................ 73
4.4 Results: TIM ......................................... 74
4.4.1 Cloning of a Phf8 - TIM targeting
construct .................................... 74
4.4.2 Targeting Phf8 in E14TG2a ES cells with the
TIM cassette ................................. 80
4.4.3 Long range PCR screening of Phf8 TIM
candidates ................................... 81
4.5 Discussion: TIM ...................................... 81
4.5.1 Cloning of the TIM cassette .................. 82
4.5.2 Targeting with the TIM cassette is not
a promoterless strategy ...................... 82
5 Novel applications for recombineering with RecE and RecT ... 84
5.1 Introduction ......................................... 84
5.2 Results: One-step generation of a targeting
construct ............................................ 86
5.2.1 E.coli strains for 3PCR recombineering ....... 87
5.2.2 Cloning of a human 0ct4-EGFP targeting
construct by 3PCR recombineering ............. 88
5.3 Discussion: One-step generation of a targeting
construct ............................................ 90
5.4 Results: Direct cloning of RTPCRs .................... 91
5.4.1 Cloning of a R6K plasmid containing
the Hprt minigene ............................ 92
5.4.2 Cloning of a jarid1b expression plasmid ...... 93
5.5 Discussion: Direct cloning of RTPCRs ................. 95
6 Summary and conclusions .................................... 96
6.1 Hprt targeting with asymmetrical targeting
constructs ........................................... 96
6.1.1 Generation of the targeting constructs ....... 97
6.1.2 Hprt targeting experiments ................... 98
6.2 Novel conditional knockout cassettes ................. 99
6.2.1 The FLEX cassette ........................... 100
6.2.2 The TIM cassette ............................ 101
6.3 Novel applications for recombineering with RecE
and RecT ............................................ 102
6.3.1 The generation of an isogenic subclone for
gene targeting .............................. 103
6.3.2 One-step generation of a targeting
construct ................................... 104
6.3.3 Direct cloning of RTPCRs .................... 104
6.3.4 Outlook ..................................... 105
7 Abbreviations ............................................. 106
8 References ................................................ 108
9 Acknowledgements .......................................... 112
10 Statement of originality .................................. 113
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