Gene: 796 Name: Grm3 Family: GPCR Subfamily: Metabotropic Glutamate Accession: AH008375 GI: 6288799
Gene 796

 

Gene 796
BLASTx Output


BLASTx Output. The query sequence is the Deltagen target gene.  The top "hits" are shown with
accession number, BLAST score and probability.  The alignment of the best hit is also shown.

BLAST DATE : 3/18/2003

 

 
                                                                 Score    E
 
 
gi|6288800|gb|AAF06741.1|AF170701_1  metabotropic glutamate ...  1690   0.0  
gi|11279202|pir||JC7160  metabotropic glutamate receptor sub...  1684   0.0  
gi|400254|sp|P31422|MGR3_RAT  Metabotropic glutamate recepto...  1671   0.0  
gi|27552883|gb|AAH41407.1|  similar to glutamate receptor, m...  1642   0.0  
 
>gi|6288800|gb|AAF06741.1|AF170701_1 metabotropic glutamate receptor 3 protein [Mus musculus]
          Length = 879
 
 Score = 1690 bits (4377), Expect = 0.0
 
 Identities = 828/873 (94%), Positives = 828/873 (94%)
 Frame = +2
 
Query: 365  MKMLTRLQVLMLALFSKGFLVSLGDHNFMRREIKIEGDLVLGGLFPINEKGTGTEECGRI 544
            MKMLTRLQVLMLALFSKGFLVSLGDHNFMRREIKIEGDLVLGGLFPINEKGTGTEECGRI
Sbjct: 1    MKMLTRLQVLMLALFSKGFLVSLGDHNFMRREIKIEGDLVLGGLFPINEKGTGTEECGRI 60
 
Query: 545  NEDRGIQRLEAMLFAIDEINKDNYLLPGVKLGVHILDTCSRDTYALEQSLEFVRASLTKV 724
            NEDRGIQRLEAMLFAIDEINKDNYLLPGVKLGVHILDTCSRDTYALEQSLEFVRASLTKV
Sbjct: 61   NEDRGIQRLEAMLFAIDEINKDNYLLPGVKLGVHILDTCSRDTYALEQSLEFVRASLTKV 120
 
Query: 725  DEAEYMCPDGSYAIQENIPLLXXXXXXXXXXXXXXXXANLLRLFQIPQISYASTSAKLSD 904
            DEAEYMCPDGSYAIQENIPLL                ANLLRLFQIPQISYASTSAKLSD
Sbjct: 121  DEAEYMCPDGSYAIQENIPLLIAGVIGGSYSSVSIQVANLLRLFQIPQISYASTSAKLSD 180
 
Query: 905  KSRYDYFARTVPPDFYQAKAMAEILRYFNWTYVSTVASEGDYGETGIEAFEQEARLRNIC 1084
            KSRYDYFARTVPPDFYQAKAMAEILRYFNWTYVSTVASEGDYGETGIEAFEQEARLRNIC
Sbjct: 181  KSRYDYFARTVPPDFYQAKAMAEILRYFNWTYVSTVASEGDYGETGIEAFEQEARLRNIC 240
 
Query: 1085 IATAEKVGRSNIRKSYDSVIRELLQKPNARVVVLFMRSDDSRELIAAASRVNASFTWVAS 1264
            IATAEKVGRSNIRKSYDSVIRELLQKPNARVVVLFMRSDDSRELIAAASRVNASFTWVAS
Sbjct: 241  IATAEKVGRSNIRKSYDSVIRELLQKPNARVVVLFMRSDDSRELIAAASRVNASFTWVAS 300
 
Query: 1265 DGWGAQESIVKGSEHVAYGAITLELASHPVRQFDRYFQSLNPYNNHRNPWFRDFWEQKFQ 1444
            DGWGAQESIVKGSEHVAYGAITLELASHPVRQFDRYFQSLNPYNNHRNPWFRDFWEQKFQ
Sbjct: 301  DGWGAQESIVKGSEHVAYGAITLELASHPVRQFDRYFQSLNPYNNHRNPWFRDFWEQKFQ 360
 
Query: 1445 CSLQNKRNHRQICDKHLAIDSSNYEQESKIMFVVNAVYAMAHALHKMQRTLCPNTTKLCD 1624
            CSLQNKRNHRQICDKHLAIDSSNYEQESKIMFVVNAVYAMAHALHKMQRTLCPNTTKLCD
Sbjct: 361  CSLQNKRNHRQICDKHLAIDSSNYEQESKIMFVVNAVYAMAHALHKMQRTLCPNTTKLCD 420
 
Query: 1625 AMXXXXXXXXXXXXXXXXNFTAPFNPNKGADSIVKFDTYGDGMGRYNVFNFQHIGGKYSY 1804
            AM                NFTAPFNPNKGADSIVKFDTYGDGMGRYNVFNFQHIGGKYSY
Sbjct: 421  AMKILDGKKLYKDYLLKINFTAPFNPNKGADSIVKFDTYGDGMGRYNVFNFQHIGGKYSY 480
 
Query: 1805 LKVGHWAETLYLDVDSIHWSRNSVPTSQCSDPCAPNEMKNMQPGDVCCWICIPCEPYEYL 1984
            LKVGHWAETLYLDVDSIHWSRNSVPTSQCSDPCAPNEMKNMQPGDVCCWICIPCEPYEYL
Sbjct: 481  LKVGHWAETLYLDVDSIHWSRNSVPTSQCSDPCAPNEMKNMQPGDVCCWICIPCEPYEYL 540
 
Query: 1985 VDEFTCMDCGPGQWPTADLSGCYNLPEDYIRWEDAWAIGPVTIACLGFMCTCIVITVFIK 2164
            VDEFTCMDCGPGQWPTADLSGCYNLPEDYIRWEDAWAIGPVTIACLGFMCTCIVITVFIK
Sbjct: 541  VDEFTCMDCGPGQWPTADLSGCYNLPEDYIRWEDAWAIGPVTIACLGFMCTCIVITVFIK 600
 
Query: 2165 HNNTPLVKASGRELCYILLFGVSLSYCMTFFFIAKPSPVICALRRLGLGTSFAICYSALL 2344
            HNNTPLVKASGRELCYILLFGVSLSYCMTFFFIAKPSPVICALRRLGLGTSFAICYSALL
Sbjct: 601  HNNTPLVKASGRELCYILLFGVSLSYCMTFFFIAKPSPVICALRRLGLGTSFAICYSALL 660
 
Query: 2345 TKTNCIARIFDGVKNGAQRPKFISPSSQVFICLGLILVQIVMVSVWLILETPGTRRYTLP 2524
            TKTNCIARIFDGVKNGAQRPKFISPSSQVFICLGLILVQIVMVSVWLILETPGTRRYTLP
Sbjct: 661  TKTNCIARIFDGVKNGAQRPKFISPSSQVFICLGLILVQIVMVSVWLILETPGTRRYTLP 720
 
Query: 2525 EKRETVILKCNVKDSSMLISLTYDVVLVILCTVYAFKTRKCPENFNEAKFIGFTMYTTCI 2704
            EKRETVILKCNVKDSSMLISLTYDVVLVILCTVYAFKTRKCPENFNEAKFIGFTMYTTCI
Sbjct: 721  EKRETVILKCNVKDSSMLISLTYDVVLVILCTVYAFKTRKCPENFNEAKFIGFTMYTTCI 780
 
Query: 2705 IWLAFLPIFYVTSSDYRVQTTTMCISVSLSGFVVLGCLFAPKVHIVLFQPQKNVVTHRLH 2884
            IWLAFLPIFYVTSSDYRVQTTTMCISVSLSGFVVLGCLFAPKVHIVLFQPQKNVVTHRLH
Sbjct: 781  IWLAFLPIFYVTSSDYRVQTTTMCISVSLSGFVVLGCLFAPKVHIVLFQPQKNVVTHRLH 840
 
Query: 2885 LNRFSVSGXXXXXXXXXXXXXVPTVCNGREVLD 2983
            LNRFSVSG             VPTVCNGREVLD
Sbjct: 841  LNRFSVSGTATTYSQSSASTYVPTVCNGREVLD 873
 
 

 


 

Construct

Gene:

796

 

GI Number(s):

6288799

ES Cell Line:

7892

 

 

Accession Number:

AH008375

 

 

Family:

GPCR

 

 

Subfamily:

Metabotropic Glutamate

 

 

Species:

Mouse

 

 

 

Sequence Type:

full length cDNA

 

 

 

 

 


Sequence Length:

3530 bp

 

red underlined text = deleted in gene sequence

Sequence Deleted:

from base 1734 to base 2755

 

black text = gene sequence flanking deleted sequence



GGGCGGGGGCAGGGGGCACTGTGACAGGAAGCTTCGCGCACAAGTTGGCCATTTCAGGGG
CAAAATAAGTTCTCCCTTGGATTTAGAAAGGACAAAGGCAGCAAGCTCCCTCTTTTGTGT
CGGATGAAGAGGACCAACCATGAGCCAGAGCTTGGGTGCAGGCTCACCGCTGCCGCTACC
ACCGCGGTCAGCTTCAGTTCCTGCCAGGAGTTGACCGTGCGAGGAATTCAGTGACGGGCT
CTTTTAGCCCTATCCTCCCTTATCTGAAGGACAGACCAAAGATCCTACTTGGAAATGAGA
GGAGACTAGCAGGAAACATTGGACCCACTCAGTGATATCTCCAAGAGATACAGAACCAGG
ATTCATGAAGATGTTGACAAGACTACAAGTTCTTATGTTAGCTTTGTTTTCAAAGGGATT
TTTAGTCTCTTTAGGAGATCACAACTTTATGAGGAGAGAAATTAAAATAGAAGGAGACCT
TGTTTTAGGGGGCTTATTTCCTATTAATGAAAAAGGCACTGGAACTGAAGAGTGTGGACG
AATCAATGAAGACAGAGGTATCCAACGCCTGGAGGCCATGTTGTTTGCCATTGATGAAAT
CAACAAAGACAATTACTTGCTTCCAGGAGTGAAGCTGGGGGTTCACATTTTGGATACATG
TTCAAGAGACACCTATGCATTAGAGCAGTCACTGGAGTTTGTCAGAGCATCGTTGACTAA
AGTGGATGAAGCTGAATATATGTGTCCTGATGGATCATATGCTATTCAAGAAAACATCCC
ACTACTCATTGCAGGAGTCATTGGCGGTTCGTACAGCAGTGTTTCCATACAGGTGGCAAA
CCTGCTGAGGCTCTTCCAGATCCCTCAGATAAGCTACGCCTCCACCAGTGCCAAACTCAG
CGACAAATCGCGCTATGATTATTTTGCCAGGACCGTGCCCCCTGACTTCTACCAGGCCAA
AGCCATGGCCGAGATCTTGCGCTACTTTAACTGGACCTATGTGTCCACTGTTGCCTCTGA
AGGTGACTATGGGGAGACAGGGATTGAGGCCTTCGAGCAGGAAGCAAGGCTACGCAACAT
CTGCATCGCCACTGCTGAAAAGGTGGGGCGCTCCAACATCCGCAAGTCCTACGACAGCGT
GATCCGTGAGCTCCTGCAGAAACCTAACGCGCGAGTTGTGGTCCTGTTCATGCGCAGTGA
TGACTCACGAGAGTTGATCGCTGCAGCCAGCCGCGTGAATGCTTCCTTCACCTGGGTGGC
CAGCGATGGCTGGGGTGCACAGGAGAGCATTGTCAAGGGCAGTGAGCACGTCGCCTATGG
AGCCATCACCCTGGAGCTGGCGTCCCACCCTGTTCGTCAGTTTGATCGCTACTTCCAGAG
CCTCAACCCCTACAACAATCATCGTAACCCCTGGTTCCGAGACTTCTGGGAGCAGAAGTT
CCAGTGCAGCCTCCAGAACAAGAGAAACCACAGACAGATTTGTGACAAGCACCTGGCCAT
TGACAGCAGCAACTATGAACAAGAATCCAAGATCATGTTTGTGGTGAATGCAGTGTATGC
CATGGCGCATGCGCTGCACAAAATGCAACGCACCCTCTGTCCCAACACCACCAAGCTCTG
TGATGCAATGAAGATCCTGGATGGAAAGAAGTTGTACAAAGATTATTTGCTGAAAATCAA
CTTCACAGCTCCATTCAACCCAAATAAAGGAGCAGACAGCATTGTGAAGTTTG
ACACTTA
CGGAGACGGGATGGGAAGATACAACGTGTTCAACTTCCAGCATATAGGTGGAAAGTATTC
CTACTTAAAAGTTGGCCACTGGGCAGAAACTTTATATCTAGATGTGGACTCTATTCATTG
GTCCCGGAACTCAGTCCCCACTTCCCAGTGCAGTGATCCCTGTGCCCCCAATGAAATGAA
AAACATGCAGCCAGGAGATGTTTGCTGCTGGATCTGCATCCCATGTGAGCCCTATGAATA
CCTGGTTGATGAGTTCACCTGCATGGATTGTGGCCCTGGCCAGTGGCCCACTGCAGACCT
ATCTGGATGCTACAACCTTCCAGAGGATTACATCAGGTGGGAAGATGCCTGGGCAATAGG
CCCAGTCACTATTGCCTGCCTGGGTTTTATGTGTACATGCATAGTCATAACTGTTTTTAT
CAAGCACAACAACACACCCTTGGTCAAAGCATCAGGCCGAGAACTCTGCTACATCTTGTT
ATTTGGAGTTAGCCTGTCCTATTGCATGACATTCTTCTTCATTGCTAAGCCATCGCCTGT
CATCTGTGCATTGCGCCGACTTGGGCTTGGGACCTCCTTTGCCATCTGTTATTCAGCTCT
CCTGACCAAGACAAACTGCATCGCTCGCATCTTTGATGGGGTCAAGAATGGCGCTCAGAG
GCCAAAATTCATCAGCCCCAGTTCTCAGGTTTTTATCTGCCTGGGTTTGATACTGGTGCA
AATTGTGATGGTGTCTGTGTGGCTTATCTTGGAGACTCCAGGTACTAGAAGATACACCCT
GCCAGAGAAGCGGGAAACAGTCATCCTAAAATGCAATGTCAAAGATTCCAGCATGTTGAT
CTCTCTGACCTATGACGTGGTTCTGGTGATTCTATGCACTGTGTATGCCTTCAAAACAAG
GAAGTGTCCTGAAAACTTCAATGAAGCCAAGTTCATAGGCTTCACCATGTACACCACCTG
CATCATCTGGTTGGCATTCCTCCCTATATTTTATGTGACATCAAGTGACTACAGA
GTGCA
GACGACAACAATGTGCATCTCCGTTAGCTTGAGTGGTTTCGTGGTCTTGGGCTGTTTGTT
TGCCCCCAAGGTGCACATTGTCCTGTTCCAACCCCAGAAGAATGTGGTCACACACAGACT
TCACCTCAACAGGTTCAGTGTCAGTGGAACTGCGACCACATATTCTCAGTCCTCTGCAAG
CACATATGTCCCGACAGTGTGCAACGGAAGGGAAGTCCTGGACTCCACCACCTCATCTCT
GTGATTGTGACTCGCAGTTCAGTTCTGTTTATAGGCTGTTAGACGAAGTGCTCACATGCT
GCTCCAGAATATGGAAACAGAACAAAAACAACAACCCTAGTACCTTTTTTTTTTTTTTTT
TTAGAAACAGTAAAATAAATTATTTTTGAGGACTGTATGGTATATAGTGATGTGCTACAA
CTTTCTAGGCTGATTTTAGTGCCCCTATTAACAGCCCCTCCCCAGGACATGGAAATAACC
ATTGTTTACAGAGCTGAGCATTGGTGACAGGGTCTGACAGGGTCAGTCTACTAAAAGAAA
ACAAAACAAAACAAAAAAACAAAAACAAAAAAATAATACAGTGGCAATATTAGGTAACCT
TTTTCCTATGAAGTCTTTTGTAGGTTCTTGTTGTAACTAATATAGGGTGAGTTTCTACGT
TGTATATTAAAGTTACATTGCGTGTAACAGAACGTTTTTCTCAGTACAAAATAAAATGCA
TCTGTATTAATGTAAAGATAACCAGAATAAAACCTTCAAGGTTTTCAAGC




Targeting Vector* (genomic sequence)

    LacZ-Neo

Construct Number:  14295

Arm Length:
5':  4  kb
3':  6  kb


  * Not drawn to scale



 


 

5 ' PCR Reconfirmation

Gene:

796

ES Line:

7892

Gene Specific Oligo (GS):
ACACAGATATTCTCACTGGGACCTGG

62064



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

4125bp

4135bp

4700bp

5167bp

4000bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

62064 + NEO1 (11193)

4125

3

62064 + NEO2 (11192)

4135

4

62064 + NEO3 (2416)

4700

5

DNA Sample Control

5167

6

Positive Control

4000

 


 

3 ' PCR Reconfirmation

Gene:

796

ES Line:

7892

Gene Specific Oligo (GS):
AGACCACGAAACCACTCAAGCTAACG

62068



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

6000bp

5950bp

5250bp

5167bp

5000bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

62068 + NEO1 (1431)

6000

3

62068 + NEO2 (1432)

5950

4

62068 + NEO3 (1170)

5250

5

DNA Sample Control

5167

6

Positive Control

5000

 


 

Legend : Genomic DNA from the recombinant ES line was assayed for homologous recombination using polymerase chain reactions (PCRs). Amplified DNA fragments were visualized by ethidium bromide staining following agarose gel electrophoresis, as shown in the gel image on the left. The test PCRs (a set of three lanes) employed the gene-specific (GS) primer, which lies outside of and adjacent to the targeting vector arm, paired in succession with one of three primers in the insertion fragment. The "DNA sample control" employed a primer pair intended to amplify a fragment from a non-targeted genomic locus. The "positive control" employed the GS primer paired with a primer at the other end of the arm. The table below the gel image lists the primers (numbered) used in each PCR and the expected product sizes, in base pairs (bp).  The diagrams to the right depict the anticipated gel image as well as the relative positions of the PCR primers.

 

 


 

5 ' Southern Blot Confirmation

Gene:

796

ES Line:

7892

Construct:

14295

Blot Number:

5617

Gene Side:

5'

 

 

MW Marker

Lane

ES Line

Enzyme

Yes

1

 

 

 

2

Parent 1

Bam HI

 

3

Parent 2

Bam HI

 

4

7892

Bam HI

 

Legend : Genomic DNA isolated from the indicated ES lines was digested with the indicated restriction enzymes (determined to cut outside of the construct arms).  The DNA was analyzed by Southern hybridization, probing with a radiolabeled DNA fragment that hybridizes outside of and adjacent to the construct arm.  The parent ES lines (negative controls) showed bands representing the endogenous (wild-type) Gene #796 allele.  In contrast, the ES#7892  line showed an additional band representing the targeted allele from the expected homologous recombination event.  
Note: All lanes represent data from the same blot.

 


 

Genotyping: Gene 796 (F2 Mice)



 

W = wild-type

H = heterozygote

K = homozygote



A) Scheme:

B) Expected Result:

 

C) Oligo Information:

Type

ID

Sequence

Multiplex Conc.

GS(E1)

68084

GTATTCATAGGGCTCACATGGGATG

400

GS(E,T)

68083

TAAAGGAGCAGACAGCATTGTGAAG

400

NEO(T)

45995

GACGTTGTTTGTCTTCAAGAAGCTTC

400

 

D) Reaction Conditions:

Allele

Expected Size

Oligos to use

Targeted

480

oligo 45995 + oligo 68083

Endogenous

277

oligo 68084 + oligo 68083