Relación de la nematofauna y las variables ambientales

A targeting vector called pTBPNS3, produced and tested by Brookes et a/., (1993), was obtained and would be used to create the targeting construct (Figure 4.2). The targeting vector contained a neomycin (neo) cassette for positive selection and the Herpes Simplex Virus thymidine kinase (HSVtk) cassette for negative selection. The aim was to clone fragments of the DSC2 gene on either side of a neomycin cassette, ensuring a disruption at exon 10. The HSVtk gene would remain close to the homologous sequences. The cloning strategy was also designed such that one o f the homologous fragments would be small enough to amplify by PGR when screening for

recombinant ES clones. In addition, it was important that the gene was disrupted within a unit codon so that if the neomycin gene was somehow spliced out, then the mRNA transcript would still be incomplete.

The cloning strategy was carried out as illustrated in Figure 4.3. Digestion o f the mouse genomic clone with Not\ and Nhel restriction endonuclease enzymes resulted in a 1.2 kb fragment with Notl and Nhel 5’ overhanging cohesive ends. The cohesive ends were compatible with the Notl and Xbal sites on the left hand side o f the neomycin gene. Cloning this fragment into the Notl and Xbal sites resulted in the loss of the Nhel restriction site with the cloned fragment having the 5’ end o f the fragment nearest the Notl site. This was confirmed by restriction digest which also showed the presence o f an extra Sail site which was present in the vector arm of the X clone (Figure 4.4A).

Figure 4.1 Restriction Digest of a mouse DSC2 genomic clone

A Restriction digest of X FIX II genomic clone to size the insert. The left arm of the vector is 20 kb and the right arm is 9 kb.

Lane 1 Notl digest Lane 2 Sail digest Lane 3 Nhel digest Lane 4 Notl + Sail digest Lane 5 Notl + Nhel digest

B Restriction digest o f X clone in Lane M indicates 1 kb marker ladder Lane 1 Notl digest

Lane 2 Sail digest Lane 3 BamRl digest Lane 4 Hindlll digest Lane 5 Sacl digest

Lane 6 BamRl + Sail digest Lane 7 BamRl + Notl digest Lane 8 Hindlll + Sail digest Lane 9 Hindlll + Notl digest Lane 10 Sacl + Notl digest Lane 11 BamRl + Nhel digest Lane 12 Hindlll + Nhel digest Lane 13 Undigested DNA

1 2 3 4 5 20 kb — 14.5 k b - 9 k b - 1.6 k b - 1 k b -

B

Figure 4.2 Targeting Vector used for the DSC2 construct and map of DSC2 clone

Above:

The vector designed by Brookes et al., (1993) contains a neomycin and the HSVtk cassette on a pBluescribe backbone. The leucine (LeuII) gene is available for use in yeast. On each side of the neomycin cassette are cloning sites containing rare restriction sites. The size o f the plasmid is 8.3 kb. The Notl sites will be used to linearise the vector.

Below:

Map o f X FIXII clone containing mouse DSC2.

S - Sall\ B - Bam Rl’, N - Notl’, Sa - Sacl’, Nh - Nhel’, No Clal sites in the insert (data not shown). The clone is thought to begin within intron 9. Notl and Sail sites flank the insert.

N X Sp Bg Nr SL \ / Sm B Xh H \ / H S V tk PTBP N S3 H - H i n d l U Xh - X h o I B - B a m H I Sm - S m a I SL - S a l I Nr - N r u I Bg - B g l I Sp - S p h I X - X ba [ N - N o t l N S

JLJ

H

The restriction digest o f the genomic clone revealed that no other restriction sites were compatible with the sites present in the second cloning site o f the targeting vector. However, whilst determining the exon/intron boundaries of the mouse DSC2 gene, a 3.6 kb fragment had been isolated by PCR and cloned into a pMOS T-vector (Amersham) for sequencing. Restriction digest analysis of this cloned fragment showed the 5’ orientation o f the insert as well as the absence of RawHI or Xhol sites in the cloned insert. This fragment could therefore be cloned into the BamYil and Xhol

sites present in the second cloning site o f the targeting construct using the sites available from the multiple cloning site of the pMOS T-vector. BamYYl and Sail

restriction sites flanked the 3.6 kb cloned insert and were used to clone the fragment into the BamYll and Xhol sites in the targeting vector, eliminating the Sail and Xhol

sites. The 5’ end o f this 3.6 kb fragment was nearest the BamYll site. After ligating the 3.6 kb fragment into the targeting construct, bacterial cells were transformed and colonies were picked. The clones were grown in 3 ml LBroth and the DNA was isolated and digested with Ndel enzyme (Figure 4.4B). During the ligation o f the 3.6 kb fragment, some o f the other restriction sites present in the pMOS T-vector were also cloned into the targeting construct including Ndel. This allowed the easy identification o f the correctly cloned fragments.

Figure 4.3 Cloning Strategy used to create the DSC2 targeting construct

The illustration in grey represents the mouse DSC2 X genomic clone showing the Notl

and Nhel restriction sites. 19, 110 and 111 indicate mouse introns 9, 10 and 11 respectively (not to scale). The 1.2 kb fragment isolated from the Notl-Nhel digest was cloned into the Notl-Xbal sites in the targeting vector. Primers RSB248 and RSB 251 were used to generate the 3.6 kb PCR fragment which was subsequently cloned into the pMOS T-vector (Amersham). The cloning sites from this vector were used to clone the PCR product into the targeting vector. Sail is compatible with Xhol but result in the loss of both restriction sites. The targeting construct was sequenced to confirm the presence and orientation of both inserts (data not shown). The targeting construct, now complete was 13.1 kb in size and was renamed VKSl.

N o t l i N h e l 19 i RSB 248

110

111

Figure 4.4 Restriction digest of cloned DSC2 fragments

A Digests o f pTBPNS3 with and without cloned fragments, and also o f 3.6 kb DSC2 fragment cloned into the pMOS T-vector.

Lane M indicates 1 kb marker ladder

Lane 1 Uncut pTBPNS3 targeting vector Lane 2 pTBPNS3 Ba/wHI digest

Lane 3 pTBPNS3 Notl digest

Lane 4 pTBPNS3 with cloned 1.2 kb fragment, uncut DNA Lane 5 pTBPNS3 with cloned 1.2 kb fragment, BamYll digest Lane 6 pTBPNS3 with cloned 1.2 kb fragment, Notl digest Lane 7 pTBPNS3 with cloned 1.2 kb fragment, Notl + Sail digest Lane 8 3.6 kb fragment in pMOS T-vector, Sacl + Xbal digest Lane 9 3.6 kb fragment in pMOS T-vector, BamY{\. + Sail digest Lane 10 3.6 kb fragment in pMOS T-vector, Hinâlll + BamYH digest Lane 11 3.6 kb fragment in pMOS T-vector, uncut DNA

B Minipreps to check for the presence of the 3.6 kb fragment, in targeting vector.

Lane M indicates 1 kb marker ladder

Lanes 1-12 indicate 12 different clones digested with Ndel enzyme. Clones 3 ,5 ,8 and