Gene: 2093 Name: Hcrtr1 Family: GPCR Subfamily: Orphan GPCR Accession: XM_131679 GI: 20845117
Gene 2093

 

Gene 2093
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 : 3/13/2003

 

                                                                         Score    E
 
 
gi|6981018|ref|NP_037196.1|  hypocretin receptor 1; Hypocret...   541   e-153
gi|17380166|sp|P58307|OX1R_MOUSE  Orexin receptor type 1 (Ox...   524   e-148
gi|11055243|gb|AAG28020.1|  hypocretin receptor-1 [Homo sapi...   515   e-146
gi|4557637|ref|NP_001516.1|  orexin receptor 1 [Homo sapiens...   513   e-145
 
>gi|6981018|ref|NP_037196.1| hypocretin receptor 1; Hypocretin (orexin) receptor 1 [Rattus
           norvegicus]
 gi|6225809|sp|P56718|OX1R_RAT Orexin receptor type 1 (Ox1r) (Hypocretin receptor type 1)
 gi|2897126|gb|AAC40041.1| orexin receptor-1; OX1R; G protein-coupled receptor [Rattus
           norvegicus]
          Length = 416
 
 Score =  541 bits (1393)Expect = e-153
 
 Identities = 267/272 (98%)Positives = 267/272 (98%)
 Frame = +2
 
Query: 2   VLTLSFIALDRWYAICHPLLFKSTARRARGSILGIWAVSLAVMVPQAAVMECSSVLPELA 181
           VLTLSFIALDRWYAICHPLLFKSTARRARGSILGIWAVSLAVMVPQAAVMECSSVLPELA
Sbjct: 134 VLTLSFIALDRWYAICHPLLFKSTARRARGSILGIWAVSLAVMVPQAAVMECSSVLPELA 193
 
Query: 182 NRTRLFSVCDEHWADELYPKIYHSCFFIVTYLAPLGLMAMAYFQIFRKLWGRQIPGTTSA 361
           NRTRLFSVCDE WADELYPKIYHSCFF VTYLAPLGLM MAYFQIFRKLWG QIPGTTSA
Sbjct: 194 NRTRLFSVCDERWADELYPKIYHSCFFFVTYLAPLGLMGMAYFQIFRKLWGPQIPGTTSA 253
 
Query: 362 LVRNWKRPSEQLEAQHQGLCTEPQPRARAFLAEVKQMRARRKTAKMLMVVLLVFALCYLP 541
           LVRNWKRPSEQLEAQHQGLCTEPQPRARAFLAEVKQMRARRKTAKMLMVVLLVFALCYLP
Sbjct: 254 LVRNWKRPSEQLEAQHQGLCTEPQPRARAFLAEVKQMRARRKTAKMLMVVLLVFALCYLP 313
 
Query: 542 ISVLNVLKRVFGMFRQASDREAVYACFTFSHWLVYANSAANPIIYNFLSGKFREQFKAAF 721
           ISVLNVLKRVFGMFRQASDREAVYACFTFSHWLVYANSAANPIIYNFLSGKFREQFKAAF
Sbjct: 314 ISVLNVLKRVFGMFRQASDREAVYACFTFSHWLVYANSAANPIIYNFLSGKFREQFKAAF 373
 
Query: 722 SCCLPGLGPGSSARHKSLSLQSRCSVSKVSEH 817
           SCCLPGLGP SSARHKSLSLQSRCSVSKVSEH
Sbjct: 374 SCCLPGLGPSSSARHKSLSLQSRCSVSKVSEH 405
 
 
 

 


 

Construct

Gene:

2093

 

GI Number(s):

20845117

ES Cell Line:

5353

 

 

Accession Number:

XM_131679

 

 

Family:

GPCR

 

 

Subfamily:

Orphan GPCR

 

 

Species:

Mouse

 

 

 

Sequence Type:

full length cDNA

 

 

 

 

 


Sequence Length:

1130 bp

 

red underlined text = deleted in gene sequence

Sequence Deleted:

from base 643 to base 652

 

black text = gene sequence flanking deleted sequence



AGTGCTGACTCTCAGCTTCATCGCCCTGGACCGCTGGTATGCCATCTGCCACCCACTGTT
GTTCAAGAGCACAGCTCGCCGTGCCCGTGGCTCCATCCTGGGCATCTGGGCTGTGTCGCT
GGCTGTCATGGTGCCCCAGGCTGCTGTCATGGAGTGCAGCAGCGTGCTGCCTGAGCTAGC
CAATCGCACCCGGCTCTTCTCTGTCTGTGATGAGCACTGGGCAGATGAACTCTACCCCAA
GATCTATCACAGCTGCTTTTTCATTGTCACCTACCTGGCCCCACTGGGCCTCATGGCTAT
GGCCTATTTCCAGATCTTCCGCAAGCTCTGGGGCCGCCAGATCCCTGGTACCACATCAGC
CTTGGTGCGGAACTGGAAACGGCCCTCGGAACAACTGGAGGCTCAGCACCAGGGCCTCTG
TACAGAGCCCCAGCCCCGGGCCCGAGCCTTCCTGGCTGAGGTGAAGCAGATGCGAGCTCG
GAGGAAGACGGCTAAGATGCTGATGGTAGTCCTGCTGGTTTTTGCACTCTGTTATCTGCC
CATCAGTGTCCTCAATGTCCTTAAGAGAGTGTTCGGGATGTTCCGCCAAGCCAGCGACCG
GGAAGCCGTCTACGCCTGCTTCACCTTCTCCCACTGGCTAGT
GTACGCCAACAGTGCCGC
CAACCCTATCATCTACAACTTCCTCAGTGGCAAATTCCGGGAGCAGTTCAAGGCTGCCTT
CTCCTGCTGCCTGCCTGGTCTGGGTCCCGGCTCCTCTGCCAGACACAAGTCCTTGTCCTT
GCAGAGCCGCTGCTCCGTCTCCAAGGTCTCTGAGCATGTCGTGCTGACCACCGTCACTAC
CGTGCTGTCCTGAGTGGGGCCGGCCCAGGCTCTGGGAGACCTGGTCCTTTGCCTGGGGTC
CACCCCAGCAGTCACAGGAAGACTGCTGCGGTTCAGTGAGAGGCTGTGCGATGCCAGTCC
TGGTTTTCTGCCAGAGTCATCCCCTCTCTGAGGTTGTGTCCCCTAAGCTGGGTGGATAGG
GGCTTAAGCTGCTCAAGAAAGCAGTGTTGCAAACCGTCTAGTGCTGTGGGCGTGGTTACT
GTCATCCCTGTCTCATATGGCTCTACCCTAGAGTGTCACTTCACCCTATC




Targeting Vector* (genomic sequence)

    LacZ-Neo

Construct Number:  10212

Arm Length:
5':  3  kb
3':  5  kb


  * Not drawn to scale



 


 

5 ' PCR Reconfirmation

Gene:

2093

ES Line:

5353

Gene Specific Oligo (GS):
GCCTATTTCCAGATCTTCCGCAAGC

35566



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

3125bp

3135bp

3700bp

5167bp

3000bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

35566 + NEO1 (11193)

3125

3

35566 + NEO2 (11192)

3135

4

35566 + NEO3 (2416)

3700

5

DNA Sample Control

5167

6

Positive Control

3000

 


 

3 ' PCR Reconfirmation

Gene:

2093

ES Line:

5353

Gene Specific Oligo (GS):
CCCCAAGACCTCTCACAGTGTGGTC

37962



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

5500bp

5450bp

4750bp

5167bp

4500bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

37962 + NEO1 (1431)

5500

3

37962 + NEO2 (1432)

5450

4

37962 + NEO3 (1170)

4750

5

DNA Sample Control

5167

6

Positive Control

4500

 


 

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.

 

 


 

3 ' Southern Blot Confirmation

Gene:

2093

ES Line:

5353

Construct:

10212

Blot Number:

4899

Gene Side:

3'

 

 

MW Marker

Lane

ES Line

Enzyme

 

1

Parent 1

Hind III

 

2

Parent 2

Hind III

 

3

5353

Hind III

 

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 #2093 allele.  In contrast, the ES#5353  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 2093 (F2 Mice)



 

W = wild-type

H = heterozygote

K = homozygote



A) Scheme:

B) Expected Result:

 

C) Oligo Information:

Type

ID

Sequence

Multiplex Conc.

GS(E,T)

49859

TCTCACTGAGCATGCTCCCTGGGTG

400

NEO(T)

3195

GGGCCAGCTCATTCCTCCCACTCAT

400

GS(E1)

49858

AGAGTGTTCGGGATGTTCCGCCAAG

400

 

D) Reaction Conditions:

Allele

Expected Size

Oligos to use

Targeted

538

oligo 3195 + oligo 49859

Endogenous

379

oligo 49858 + oligo 49859